bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of three-dimensional replication patterns during the s-phase, analysed by double labelling of dna and confocal microscopy
Authors: Manders E. M. M. , Stap J. , Brakenhoff G. J. , Driel R. Van , Aten J. A.
Published: April 2021

The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.
DNA replication in chloroplasts
Authors: Heinhorst Sabine , Cannon Gordon C.
Published: April 2021

Chloroplasts contain multiple copies of a DNA molecule (the plastome) that encodes many of the gene products required to perform photosynthesis. The plastome is replicated by nuclear-encoded proteins and its copy number seems to be highly regulated by the cell in a tissue-specific and developmental manner. Our understanding of the biochemical mechanism by which the plastome is replicated and the molecular basis for its regulation is limited. In this commentary we review our present understanding of chloroplast DNA replication and examine current efforts to elucidate its mechanism at a molecular level.
Thermotolerance in mammalian cells protein denaturation and aggregation, and stress proteins
Authors: Kampinga Harm H.
Published: April 2021

Cells that have been pre-exposed to thermal stress can acquire a transient resistance against the killing effect of a subsequent thermal stress. The cause for this phenomenon, called thermotolerance, seems to be an enhanced resistance of proteins against thermal denaturation and aggregation. This resistance can be expressed as an attenuation of damage formation (less initial damage) or as a better repair of the protein damage (facilitated recovery). Heat Shock (or better, Stress) Proteins (HSPs) may play a role in and even be required for thermal resistance. However, rather than stress-induced enhanced synthesis and elevated total levels of HSPs per se, the concentration of, both constitutive and inducible, HSPs at and/or (re)distributed to specific subcellular sites may be the most important factor for the acquisition of thermotolerance. Specific HSPs may be involved either in damage protection or in damage repair.
Microtubule nucleating activity of centrosomes in cell-free extracts from Xenopus eggs: involvement of phosphorylation and accumulation of pericentriolar material
Authors: Ohta Kunihiro , Shiina Nobuyuki , Okumura Eiichi , Hisanaga Shin-Ichi , Kishimoto Takeo , Endo Sachiko , Gotoh Yukiko , Nishida Eisuke , Sakai Hikoichi
Published: April 2021

We have studied the regulation of microtubule nucleating activity of the centrosome using cell-free extracts from Xenopus eggs. We found that the number of microtubules per centrosome increases dramatically with time during incubation of isolated centrosomes in interphasic egg extracts prepared 20-30 minutes after electric activation of cytostatic factor (CSF)-arrested eggs. The increase in microtubule nucleation was still conspicuous even when KCl-treated centrosomes (centrosomes stripped of their microtubule nucleating activity by 1 M KCl treatment) were incubated in interphasic extracts. Electron microscopy and immunostaining by anti--tubulin and 5051 human anti-centrosome antibodies revealed that pericentriolar material (PCM) was accumulated during the increase in microtubule nucleation from centrosomes in interphasic extracts, suggesting regulation of centrosomal activity by PCM accumulation. The ability of egg extracts to activate microtubule nucleation from centrosomes was also assumed to be regulated by phosphorylation, since addition of protein kinase inhibitors into interphasic extracts totally blocked the increase in microtubule nucleation from the KCl-treated centrosome. The ability of CSF-arrested mitotic extracts to increase microtubule nucleation from KCl-treated centrosomes was 3.5-to 5-fold higher than that of interphasic extracts, while PCM accumulation in mitotic extracts seemed to be similar to that in interphasic extracts. The increase in microtubule nucleation from KCl-treated centrosomes was strikingly enhanced by the addition of purified p34cdc2/cyclin B complex to interphasic extracts, but not by MAP kinase, which is activated downstream of p34cdc2/cyclinB. These results suggest two pathways activating centrosomal activity in egg extracts: accumulation of PCM and phosphorylation mediated by p34cdc2/cyclin B.
The gene encoding the calcium binding protein calcyclin is expressed at sites of exocytosis in the mouse
Authors: Timmons Paula M. , Chan C.-T. J. , Rigby Peter W. J. , Poirier Françoise
Published: April 2021

Calcyclin is a member of the S100 family of calcium binding proteins. We have found by in situ hybridization that calcyclin transcripts are restricted to specific cell types within a limited number of mouse organs. High levels of expression in the epithelia lining the gastrointestinal, respiratory and urinary tracts, and specific localization of the transcripts to the goblet cells in the small intestine, lead us to suggest a role for calcyclin in the process of mucus secretion. In addition, calcyclin expression was detected in the corpus luteum, placenta and nerves within the gut wall, which are all sites of regulated exocytosis. We propose that this S100-like protein may be part of a calcium signalling pathway utilized in the secretion of various products by different cell types.
The membrane fusion events in degranulating guinea pig eosinophils
Authors: Lindau M. , Nüsse O. , Bennett J. , Cromwell O.
Published: April 2021

We have investigated the granule fusion events associated with exocytosis in degranulating peritoneal guinea pig eosinophils by time-resolved patch-clamp capacitance measurements using the phase detector technique. Intracellular stimulation with micromolar calcium and GTPγS induces a 2-to 3-fold capacitance increase. The main phase of the capacitance increase occurs after a delay of 2-7 minutes and is composed of well-resolved capacitance steps. The number of steps is very close to the number of crystalloid granules contained in a resting cell and the step size distribution with a peak at 9 fF is in excellent agreement with the granule size distribution determined by electron microscopy. The individual granules thus fuse sequentially with the plasma membrane. The stepwise capacitance increase is frequently preceded by an apparently continuous capacitance increase which consists of steps smaller than 4 fF, indicating exocytosis of small vesicles as distinct from crystalloid-containing granules. In some cases the time course of the opening of individual fusion pores could be recorded, and this revealed metastable conductance states below 300 pS but random fluctuations at higher conductance levels. This behaviour suggests that the small fusion pore might be a protein structure similar to an ion channel, which becomes a continuously variable lipid pore at higher conductances.In some cells a significant capacitance decrease was observed which is apparently continuous, suggesting a process of membrane uptake by endocytosis of small vesicles.
Human retinal pigment epithelial cells from different donors continuously produce a vascular endothelial cell-stimulating factor into serum-free medium
Authors: Yang Q. R. , Smets R. M. E. , Neetens A. , Berghe D. Vanden
Published: April 2021

Mitogenic activities of human retinal pigment epithelial cell-conditioned medium (HRPE-CM) with different effects, such as inhibition, stimulation or no effect, on the proliferation of vascular endothelial cells (EC) in vitro have been reported. In this study, 14 HRPE cell lines were established from normal human eyes. Human umbilical vein endothelial cells (HUVEC) in the early passages were used as target cells to detect the mitogenic activity of HRPE-CM on the growth of vascular EC. Our results confirm that HRPE cells in culture continuously synthesize and secrete HUVEC growth substance(s) into a serum-free medium. The ability of HRPE cell lines to produce this mitogen seem unrelated either to in vivo donor factors or to in vitro cell life span. Using an enzyme-linked immunosorbance assay, we demonstrated that only HRPE cell extract, not HRPECM, can be recognized by basic fibroblast growth factor (bFGF)-specific antibody, though identical bioactivities on the growth of HUVEC were found in both preparations. The active component in HRPE-CM was heat- and trypsin-sensitive, and stable at extremes of pH (2.5 to 10.0). In addition, the bioactive molecule could not pass through a Mr 30,000 cut-off membrane, suggesting that it is a fairly high molecular mass polypeptide. These observations suggest that the EC growth factor in HRPE-CM is distinct from fibroblast growth factors (FGFs).
Proliferative behaviour of high-ploidy cells in two murine tumour lines
Authors: Hoz Carmen de la , Baroja Antonio
Published: April 2021

The presence of high-ploidy cells in malignant tumours has long been documented. However, the biological significance of these cells is not known and there is a great deal of controversy over their proliferative potential. We have analysed the behaviour of these cells in two murine tumour lines, B16F10 melanoma and 3T3A31M angiosarcoma, determining their DNA content by microspectrophotometry and using time-lapse film studies. We have found a discrepancy between the presence of high-ploidy cells in metaphase and the absence of hyperploid telophases. High-ploidy metaphases may be aborted (mitotic polyploidization), prolonged in time or evolve in the form of multipolar, generally tripolar, mitoses. Our results suggest that high-ploidy cells are capable of proliferating, despite certain peculiarities in their cell cycle, and constitute a tumour subpopulation whose role in neoplasia merits further study.
Immortalization of polarized rat retinal pigment epithelium
Authors: Nabi Ivan R. , Mathews Abraham P. , Cohen-Gould Leona , Gundersen Doris , Rodriguez-Boulan Enrique
Published: April 2021

Rat retinal pigment epithelial (RPE) cells were immortalized by infection with a temperature-sensitive tsA SV40 virus and following cloning and selection for epithelial properties the polarized RPE-J cell line was obtained. At the permissive temperature of 33°C, RPE-J cells behave as an immortalized cell line. When RPE-J cells are grown on nitrocellulose filters coated with a thin layer of Matrigel in the presence of 10 8 M retinoic acid for 6 days at 33°C and then switched for 33-36 hours to the non-permissive temperature of 40°C, they acquire a differentiated polarized RPE phenotype. Under these growth conditions, RPE-J cells exhibit circumferential staining for the tight-junction protein ZO-1 and acquire a transepithelial resistance of 350 ohms cm2. Morphologically, RPE-J cells exhibit a character-istic RPE morphology with extensive apical microvilli as well as numerous dense bodies including premelanosomes and varied multilamellar structures. Ruthenium red labeling revealed the frequent basal localization of the tight junction. The cells were identified to be of rat RPE origin by their expression of the rat RPE marker RET-PE2 and their ability to phagocytose latex beads. While RPE-J cells are capable of sorting influenza and vesicular stomatitis virus to the apical and basal surfaces, respectively, the Na,K-ATPase is not polarized and the neural cell adhesion molecule, NCAM, is localized exclusively to the lateral surface. In vivo the apical surface of RPE interacts with the adjacent neural retina and the Na,K-ATPase and N-CAM are both apical; the altered polarity of these two proteins in RPE-J cells may be a consequence of the absence of apical interaction with the neural retina in culture. Previous studies of RPE have been restricted to the use of primary cultures and the RPE-J cell line should prove an excellent model system for the study of the mechanisms determining the characteristic polarity and functions of the retinal pigment epithelium.
Uptake of glutamate, not glutamine synthetase, regulates adaptation of mammalian cells to glutamine-free medium
Authors: McDermott R. H. , Butler M.
Published: April 2021

Two cell lines (McCoy and MDCK) were studied in an attempt to understand the metabolic changes associated with adaptation to glutamine-free medium (GMEM gmate). McCoy cells assumed normal growth rates after 2-3 passages in this medium whereas MDCK cells showed no growth in GMEM + gmate.

The glutamine synthetase (GS) activity of both cell lines was elevated (up to ×9) as glutamine was depleted from normal media (GMEM gmine). The high activity of GS was maintained during McCoy cell growth in GMEM + gmate. However, there was no apparent significant difference between the two cell lines in the pattern of changes of GS activity in response to glutamine.

The cellular uptake rates of glutamine and glutamate from the medium differed significantly between the two cell lines. During the adaptation of McCoy cells to GMEM gmate, the rate of glutamate uptake doubled to a value of 0.54 nmol/min per mg cell protein whereas the maximum value for MDCK cells was considerably lower (0.04 nmol/min per mg cell protein). We propose that the difference in intrinsic ability for glutamate transport accounts for the difference in growth response between the two cell lines in the glutamine-free medium.
Basement membrane and interstitial matrix components form separate matrices in heterokaryons of PYS-2 cells and fibroblasts
Authors: Laurila Pekka , Leivo Ilmo
Published: April 2021

In order to gain further understanding of the spatial organization of interstitial and basement membrane matrices, we studied the expression of the interstitial matrix protein, fibronectin, and the basement membrane protein, laminin, in heterokaryons formed by the fusion of normal fibroblasts and teratocarcinoma-derived epithelial PYS-2 cells. These heterokaryons showed various distributions of the matrix proteins depending on the proportions of the different parental cell nuclei within the cytoplasm of the cell. Heterokaryons containing equal numbers of fibroblast and PYS-2 cell nuclei showed an abundant laminin matrix subcellularly and only minor amounts of fibronectin matrix at the periphery of the cells. Similar results were obtained in heterokaryons containing an excess of epithelial cell nuclei. In heterokaryons containing an excess of fibroblast nuclei, on the other hand, laminin matrix was reduced and a fibrillar fibronectin matrix was seen also on top of the cell body. The results suggest a gene dosage-type of effect on the expression of these proteins. Furthermore, extracellular laminin and fibronectin matrices did not codistribute around the heterokaryons but the two proteins were assembled into separate structures. The lack of codistribution of fibronectin and laminin matrices in heterokaryons suggests that the molecular interactions, which determine the assembly of basement membrane and interstitial matrices in these cells are highly type-specific. Similar mechanisms may also operate in the assembly of extracellular matrices in vivo.
Tenascin expression in the mouse: in situ localization and induction in vitro by bFGF
Authors: Tucker Richard P. , Hammarback James A. , Jenrath David A. , Mackie Eleanor J. , Xu Yue
Published: April 2021

The glycoprotein tenascin is found in the extracellular matrix in regions of cell motility, cell proliferation, and tissue modelling. We have used novel tenascin cDNA probes to localize tenascin transcripts in the developing mouse and to study the regulation of tenascin expression by growth factors in vitro. At postnatal day 1 tenascin mRNAs are abundant in regions of bone and cartilage formation, as well as in the ependymal layer of the central nervous system. Previous studies have demonstrated that transforming growth factor-β type 1 (TGF-β1) can induce tenascin expression in vitro. As TGF-β1 is absent or scarce in the developing brain, it is likely that other growth factors, alone or in addition to TGF-β1, may regulate tenascin expression during development. Therefore, we have compared the effects of TGF-β1 and a growth factor that is found in both developing connective tissue and the central nervous system, basic fibroblast growth factor (bFGF), on tenascin expression in a mouse embryo fibroblast cell line (Swiss 3T3 cells). Immuno-slot blot analysis of Swiss 3T3 cell-conditioned culture medium demonstrates that bFGF is a more potent inducer of tenascin expression than TGF-β1. Furthermore, bFGF and TGF-β1 have an additive effect on levels of tenascin, but not fibronectin, in the conditioned medium. Western blots revealed that different forms of tenascin are induced by bFGF and TGF-β1: the tenascin induced by the former has a molecular mass of approximately 250 kDa, the latter induces an approximately 200 kDa form of tenascin. The induction of large tenascin by bFGF was confirmed by northern blot analysis, which revealed increased levels of an 8 kb tenascin transcript after 24 h by as little as 4 ng/ml of bFGF in serum-free medium. Thus bFGF, alone or in combination with TGF-β1, is a potential regulator of tenascin expression in vitro. bFGF may alter not only the relative abundance of tenascin and fibronectin in the extracellular matrix, but also the splice variant of tenascin expressed by a given cell type.
Characterization of the nuclear translocation of acidic fibroblast growth factor
Authors: Cao Yihai , Ekström Maria , Pettersson Ralf F.
Published: April 2021

The subcellular localization of human acidic FGF (aFGF; FGF-1) expressed to high levels by using a bacteriophage T7 RNA polymerase-driven vaccinia virus expression system was studied in BHK21 and HeLa cells. Acidic FGF was detected by immunoblotting or immunofluorescence using an affinity-purified rabbit polyclonal antibody. The nuclei of most transfected cells, but not nuclei of control cells, were strongly immunoreactive. The nuclear accumulation of aFGF was confirmed by subcellular fractionation and immunoblotting, indicating that about 50% of the expressed protein was located in the nuclei at 12 h after transfection. It has previously been reported that a putative N-terminal nuclear localization sequence (NLS) in aFGF is required for full mitogenic activity (Imamura et al., Science 249, 1567-1570, 1990). We found that deletion of the first 27 residues including the putative NLS did not prevent the nuclear translocation of aFGF in either cell type. This observation suggests that the putative NLS sequence is not essential for targeting aFGF to the cell nucleus. To analyze further the mechanism of nuclear import, purified aFGF was microinjected into the cytoplasm of growing BHK21 cells under various conditions. In chilled (4°C) or ATP-depleted cells, the injected aFGF entered the nucleus with similar efficiency to that in control cells at 37°C. This suggests that aFGF, which has a molecular mass of only 16,500, enters the cell nucleus by free diffusion, and possibly becomes trapped by binding to some nuclear structures. When added exogenously to growing BHK21 cells, aFGF was not localized to the nucleus. Instead, a punctate staining pattern in the cytosol was observed, reminiscent of that in the endosomal-lysosomal compartments. In addition, a diffuse extracellular surface-staining was evident. This result demonstrates that receptor-mediated endocytosis of aFGF does not result in its translocation to the nucleus, as has been reported for basic FGF.
A novel member of the dynamin family of GTP-binding proteins is expressed specifically in the testis
Authors: Nakata Takao , Takemura Reiko , Hirokawa Nobutaka
Published: April 2021

Dynamin is a member of a new GTPase family, which includes the mouse Mx protein, the yeast VPS1 and the Drosophila shibire gene product. A high homology with the shibire product suggests a role for dynamin in the endocytotic process, but it is expressed only in mature neurons. We identified two additional dynamin-like pro-teins in rats, by using the polymerase chain reaction with degenerate primers corresponding to the GTP-binding areas conserved between dynamin and VPS1. The full coding sequence of one of them, dynamin-2, revealed that it has 848 amino acids and has great sim-ilarity with brain dynamin and the shibire product.

Northern blot analysis and in situ hybridization revealed its expression to be specific to the seminiferous tubules in the testis. Dynamin-2 (testis type dynamin) was expressed in germ-cell-depleted testis as well, indi-cating its expression in Sertoli cells. Our data imply that a number of dynamin family proteins, which are prod-ucts of distinct genes, may play different roles specific to each cell type in the same rat.
γ-Tubulin is present in acentriolar MTOCs during early mouse development
Authors: Gueth-Hallonet Catherine , Antony Claude , Aghion Joël , Santa-Maria Angelica , Lajoie-Mazenc Isabelle , Wright Michel , Maro Bernard
Published: April 2021

γ-Tubulin, a recently discovered member of the tubulin superfamily, is a peri-centriolar component considered to be essential for microtubule nucleation. Mouse oocytes and early embryos lack centrioles until the blas-tocyst stage. Thus, early mouse embryos allowed us to study the location of γ-Tubulin in animal cells in the absence of centrioles. For this, we used an antiserum directed against a specific peptide of the γ-Tubulin sequence, which is conserved among species. This serum has been characterised both in PtK2 and mouse cells. We found that it specifically stained the spindle poles and the cytoplasmic microtubule organizing centers in metaphase II oocytes and the spindle poles in mitosis during the cleavage stages. In contrast, no interphase staining could be detected during cleavage. Since the overall level of γ-Tubulin did not decrease during inter-phase, as shown by immunoblotting experiments, this absence of staining during interphase is probably due to a cytoplasmic dispersion of γ-Tubulin. A single dot-like interphase reactivity appeared at the 32-cell stage. In parallel, electron microscopy studies allowed us to detect centrioles for the first time at the 64-cell stage. The possible roles of γ-Tubulin in microtubule nucleation and in centrosome maturation are discussed.
5-Bromo-2-deoxyuridine regulates invasiveness and expression of integrins and matrix-degrading proteinases in a differentiated hamster melanoma cell
Authors: Thomas Linda , Chan Patti Walter , Chang Sunghea , Damsky Caroline
Published: April 2021

Cell interactions with the extracellular matrix play a critical role in regulating complex processes such as ter-minal differentiation and tumor progression. In these studies we describe a melanoma cell system that should be useful in addressing the regulation of cell-matrix interactions and the roles they play in regulating differ-entiation and cell invasiveness. CS (suspension)-1 melanoma cells are relatively well differentiated: they are melanotic, responsive to melanocyte-stimulating hormone, and express TA99, a melanosome membrane differentiation marker. Their repertoire of integrin receptors for extracellular matrix ligands is limited; in particular, they lack receptors for vitronectin, account-ing for the observation that they are nonadherent when cultured in the presence of serum. CS-1 cells are non-invasive as well, and express low levels of both metal-loproteinases and activated plasminogen activators. Treatment of these cells with melanocyte-stimulating hormone causes them to increase melanin production and assume an arborized phenotype, suggesting that it promotes their further differentiation. In contrast, treatment of CS-1 with the thymidine analog 5-bromo-deoxyuridine, converts them to a highly invasive cell population (termed BCS-1) that loses its differentiated properties and responsiveness to melanocyte-stimulat-ing hormone, acquires a broad integrin repertoire (including vitronectin receptors), and expresses elevated levels of metalloproteinases and activated urokinase. From these observations and findings of others on BrdU treatment of other developmental lineages, we hypoth-esize that BrdU both suppresses differentiation and pro-motes invasiveness of CS-1 cells. The demonstrated manipulability of CS-1 cells should make them extremely useful for studying the regulation of both ter-minal differentiation and tumor progression in the melanocyte lineage.
Growth cone guidance and neuron morphology on micropatterned laminin surfaces
Authors: Clark Peter , Britland Stephen , Connolly Patricia
Published: April 2021

Neurite growth cones detect and respond to guidance cues in their local environment that determine stereo-typed pathways during development and regeneration. Micropatterns of laminin (which was found to adsorb preferentially to photolithographically defined hydro-phobic areas of micropatterns) were here used to model adhesive pathways that might influence neurite exten-sion. The responses of growth cones were determined by the degree of guidance of neurite extension and also by examining growth cone morphology. These parame-ters were found to be strongly dependent on the geom-etry of the patterned laminin, and on neuron type. Decreasing the spacing of multiple parallel tracks of laminin alternating with non-adhesive tracks, resulted in decreased guidance of chick embryo brain neurons. Single isolated 2 μm tracks strongly guided neurite extension whereas 2 μm tracks forming a 4 μm period multiple parallel pattern did not. Growth cones appear to be capable of bridging the narrow non-adhesive tracks, rendering them insensitive to the smaller period multiple parallel adhesive patterns. These observations suggest that growth cones would be unresponsive to the multiple adhesive cues such as would be presented by oriented extracellular matrix or certain axon fascicle structures, but could be guided by isolated adhesive tracks. Growth cone morphology became progressively simpler on progressively narrower single tracks. On narrow period multiple parallel tracks (which did not guide neurite extension) growth cones spanned a number of adhesive/non-adhesive tracks, and their mor-phology suggests that lamellipodial advance may be independent of the substratum by using filopodia as a scaffold. In addition to acting as guidance cues, laminin micropatterns also appeared to influence the production of primary neurites and their subsequent branching. On planar substrata, dorsal root ganglion neurons were multipolar, with highly branched neurite outgrowth whereas, on 25 μm tracks, neurite branching was reduced or absent, and neuron morphology was typi-cally bipolar. These observations indicate the precision with which growth cone advance may be controlled by substrata and suggest a role for patterned adhesiveness in neuronal morphological differentiation, but also high-light some of the limitations of growth cone sensitivity to substratum cues.
Angiogenic oligosaccharides of hyaluronan enhance the production of collagens by endothelial cells
Authors: Rooney P. , Wang M. , Kumar P. , Kumar S.
Published: April 2021

The present study demonstrates a relationship between angiogenic oligosaccharides of hyaluronan (HA) and the production of collagens during the process of angiogen-esis in vivo and in vitro. The addition of angiogenic oligosaccharides of HA to the chorioallantoic membrane of the chick embryo induced a deposition of collagen fib-rils. The treatment of sub-confluent cultures of bovine aortic endothelial cells with the same oligosaccharides (1 μg/ml) increased the uptake of [3H]proline by approx-imately 60%. SDS-polyacrylamide gel electrophoresis of treated cultures demonstrated the enhanced synthesis of type I and type VIII collagens. The production of type VIII collagen was confirmed by western blotting and immunocytochemistry using antibodies to sheep and bovine type VIII collagen. Type VIII collagen is a short chain collagen that has a high degree of homology to cartilage-specific type X collagen. The biological func-tions of type VIII and type X collagens are unknown. We have suggested that the two collagens play a role in the process of angiogenesis.
Characterisation of an epithelium-like layer of cells in the multicellular Dictyostelium discoideum slug
Authors: Fuchs Manuela , Jones Malcolm K. , Williams Keith L.
Published: April 2021

Ultrarapid freezing (RF) followed by freeze-substitution (FS) provide superior preservation of the Dictyostelium discoideum multicellular slug tissue over conventional methods of chemical fixation at room temperature. The peripheral cells of slugs prepared by RF and FS form a tight layer of flattened cells. This cell layer resembles epithelia of other multicellular organisms in that it has close junctional contact between cells associated with the extracellular matrix (ECM, slime sheath). This is the first report that clearly demonstrates the existence of such peripheral cellular specialisation in this otherwise well-studied model system. Junctional contacts between adjacent cells mean that there is no intercellular space evident between apical membranes of apposing cells, and basally the intermembraneous space between peripheral cells is less than 10 nm. By contrast, the inter-cellular space between internal cells is approximately 10–25 nm. The shape of the peripheral cells varies with their location around the slug. In the posterior prespore zone, the peripheral cells are squamous and exhibit polarity along their antero-posterior axis. In the ante-rior prestalk zone, peripheral cells are less flattened, project irregular filipodia between internal cells, and are polarised along their apical-basal axis. Colloidal gold immunocytochemistry with the markers MUD1, MUD50 and MUD62 demonstrated that the peripheral layer is formed of prestalk cells in the anterior region and ventrum, and mostly prespore cells along the dorsum. Thus, the peripheral layer, while having specific cell classes in different regions, is not differen-tiation-specific. Rather, it appears that the structure of these epithelium-like cells is influenced by interaction with molecules of the ECM (sheath).
A tau-like protein interacts with stress fibers and microtubules in human and rodent cultured cell lines
Authors: Cross Daniel , Vial Clarisa , Maccioni Ricardo B.
Published: April 2021

The cytoskeletal integrity of human and rodent cell lines was analyzed using site-directed monoclonal antibodies prepared from hybridomas. Secreting hybridomas were produced by immunizing mice with synthetic peptides from the C-terminal domain of the II-tubulin isotype, II(422-434), YQQYQDATADEQG, and the first imperfect repeat from brain tau, Tau-I(187-204), VRSKIGSTENLKHQPGGG. Two hybridomas were selected for this work: MTB6.22, an anti-idiotypic mon-oclonal antibody, which was obtained from a mouse immunized with the II-peptide and recognizes specific tubulin-binding domains on MAP-2 and tau; and Tau-I/1, which recognizes the repetitive binding sequences on tau and MAP-2. Immunoblots of cytoskeletal protein preparations from the five different tumor cell lines studied, showed the interaction of the site-directed anti-bodies MTB6.22 and Tau-I/1 with a group of proteins that co-migrate with brain tau. Immunoreactive tau components were also identified using an anti-tau mon-oclonal antibody (clone Tau-2), and several polyclonal anti-tau antibodies that interact with tau epitopes, other than those of the tubulin-binding domains. These tau-like proteins bound to a calmodulin-Sepharose affinity column and were eluted using 2 mM EGTA. Interestingly, washing the extracted cytoskeleton pellet with 5 10−3 M Ca2+for short periods of time selectively released the tau-like protein components, whilst most of the other cytoskeletal proteins remained in the pellet. On the other hand, immunofluorescence microscopy of detergent-extracted cells showed immunostaining of MAP components that appear to be co-localized in a dis-crete dot-like distribution along the stress fibers, which were revealed using rhodamine-phallacidin. Further support for the specificity of tau interaction with sites on tubulin and actin polymers was obtained with double-immunofluorescence, using the MAP-reactive monoclonal antibody MTB6.22 and a polyclonal anti-body to a tubulin peptide containing part of the tau-binding domain on tubulin.

Considering the anti-idiotypic nature of the MTB6.22 monoclonal antibody, our studies indicate that, in all the cell lines analyzed, a tau-like protein component is involved in mediating the interaction of both actin and tubulin polymers.
Expression of E- or P-cadherin is not sufficient to modify the morphology and the tumorigenic behavior of murine spindle carcinoma cells: Possible involvement of plakoglobin
Authors: Navarro Pilar , Lozano Encarnación , Cano Amparo
Published: April 2021

Transfection of E- and P-cadherin cDNA has been carried out in murine spindle carcinoma cells previously shown to be deficient in both cadherins (Navarro et al., J. Cell Biol. 115, 517-533, 1991). High levels of expression of E- or P-cadherin do not significantly affect the fibroblastic morphology of the parental spindle cells. In addition, the tumorigenic behavior of these highly malignant cells is not influenced by the ectopic expression of either cadherin. Nevertheless, a fraction of the exogenous cadherins is able to associate to detergent-insoluble components of the transfectant cells, and the expression of the exogenous E-cadherin confers Ca2+-dependent aggregation on the spindle transfectants in an in vitro assay. Immunoprecipitation analysis of the cadherin-catenin complex of the transfectants revealed that the ectopic E-cadherin associates with the ơ- and β-catenin proteins. However, the γ-catenin/plakoglobin component could not be detected in the E-cadherin immunocomplexes of the spindle transfectant cells, in contrast to the epithelial cells where the three catenins appeared to be associated with E-cadherin. The lack of association of γ-catenin is correlated with very low levels of plakoglobin in whole cell extracts of the parental spindle cells. These results indicate that the association of E-cadherin with the α- and β-catenin components is not sufficient to promote a fibroblastoid-epithelial conversion of highly malignant spindle cells. The presence of plakoglobin could be required for the proper organization of E-cadherin in the transfectant cells in order to acquire an epithelioid phenotype.
Novel structural organisation of a Mus musculus dba/2 chromosome shows a fixed position for the centromere
Authors: Mitchell Arthur R. , Nicol Linda , Malloy Patricia , Kipling David
Published: April 2021

Chromosome 1 of the inbred mouse strain DBA/2 shows an unusual polymorphism associated with its centromeric satellite DNA sequences. The minor satellite array has undergone amplification and is present as two blocks separated by major satellite sequences. Both minor satellite blocks appear to carry the sequence motif necessary for CENP-B protein binding. Despite this apparent similarity the functional centromere, as defined by the location of CREST antigens, appears to form only within the more terminal block. The two blocks also vary in that sister chromatid association only occurs with this more terminal block.
The role of isoprenylation in membrane attachment of nuclear lamins: A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties
Authors: Hennekes Hartwig , Nigg Erich A.
Published: April 2021

Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.
Identification of MYO4, a second class V myosin gene in yeast
Authors: Haarer B. K. , Petzold A. , Lillie S. H. , Brown S. S.
Published: April 2021

We have isolated a fourth myosin gene (MYO4) in yeast (Saccharomyces cerevisiae). MYO4 encodes a ∼170 kDa (1471 amino acid) class V myosin, using the classification devised by Cheney et al. (1993a; Cell Motil. Cytoskel. 24, 215-223); the motor domain is followed by a neck region containing six putative calmodulin-binding sites and a tail with a short potential ‘coiled-coil’ domain. A comparison with other myosins in GenBank reveals that Myo4 protein is most closely related to the yeast Myo2 protein, another class V myosin. Deletion of MYO4 produces no detectable phenotype, either alone or in conjunction with mutations in myo2 or other myosin genes, the actin gene, or secretory genes. However, overexpression of MYO4 or MYO2 results in several morphological abnormalities, including the formation of short strings of unseparated cells in diploid strains, or clusters of cells in haploid strains. Alterations of MYO4 or MYO2 indicate that neither the motor domains nor tails of these myosins are required to confer the over-expression phenotype, whereas the neck region may be required. Although this phenotype is similar to that seen upon MYO1 deletion, we provide evidence that the overexpression of Myo4p or Myo2p is not simply interfering with Myo1p function.
Determinants of Drosophila zw10 protein localization and function
Authors: Williams Byron C. , Goldberg Michael L.
Published: April 2021

We have examined several issues concerning how the Drosophila l(1)zw10 gene product functions to ensure proper chromosome segregation. (a) We have found that in zw10 mutant embryos and larval neuroblasts, absence of the zw10 protein has no obvious effect on either the congression of chromosomes to the metaphase plate or the morphology of the metaphase spindle, although many aberrations are observed subsequently in anaphase. This suggests that activity of the zw10 protein becomes essential at anaphase onset, a time at which the zw10 protein is redistributed to the kinetochore region of the chromosomes. (b) The zw10 protein appears to bind to kinetochores in mitotically arrested cells, eventually accumulating to high levels within the chromosome mass. Our results imply that zw10 may act as part of a novel feedback pathway that normally renders sister chromatid separation dependent upon spindle integrity. (c) The localization of zw10 protein is altered by two mitotic mutations, rough deal and abnormal anaphase resolution, that specifically disrupt anaphase. These findings indicate that the zw10 protein functions as part of a multicomponent mechanism ensuring proper chromosome segregation at the beginning of anaphase.
Intercellular communication between epithelial and fiber cells of the eye lens
Authors: Bassnett Steven , Kuszak Jerome R. , Reinisch Lou , Brown Henry G. , Beebe David C.
Published: April 2021

Results of electrical, dye-coupling and morphological studies have previously suggested that gap junctions mediate communication between the anterior epithelium of the lens and the underlying lens fiber cells. This connection is believed to permit ‘metabolic cooperation’ between these dissimilar cell types and may be of particular importance to the fiber cells, which are thought incapable of autonomous ionic homeostasis. We reinvestigated the nature of the connection between epithelial and fiber cells of the embryonic chicken lens using fluorescence confocal microscopy and freeze-fracture analysis. In contrast to earlier studies, our data provided no support for gap-junction-mediated transport from the lens epithelium to the fibers. Fluorescent dyes loaded biochemically into the lens epithelium were retained there for more than one hour. There was a decrease in epithelial fluorescence over this period, but this was not accompanied by an increase in fiber cell fluorescence. Diffusional modeling suggested that these data were inconsistent with the presence of extensive epithelium-fiber cell coupling, even if the observed decrease in epithelial fluorescence was attributed exclusively to the diffusion of dye into the fiber mass via gap junctions. Furthermore, the rate of loss of fluorescence from isolated epithelia was indistinguishable from that measured in whole lenses, suggesting that decreased epithelial fluorescence resulted from photobleaching and leakage of dye rather than diffusion, via gap junctions, into the fibers. Analysis of freeze-fracture replicas of plasma membranes at the epithelial-fiber cell interface failed to reveal evidence of gap-junction plaques, although evidence of endocytosis was abundant. These studies were done under conditions where the location of the fracture plane was unambiguous and where gap junctions could be observed in the lateral membranes of neighboring epithelial and fiber cells. Paradoxically, tracer molecules injected into the fiber mass were able to pass into the epithelium via a pathway that was not blocked by incubation at 4°C or by treatment with octanol and which excluded large (∼10 kDa) molecular mass tracers. Together with previous measurements of electrical coupling between fiber cells and epithelial cells, these data indicate the presence of a low-resistance pathway connecting these cell types that is not mediated by classical gap junctions.
The dynein genes of paramecium tetraurelia: sequences adjacent to the catalytic p-loop identify cytoplasmic and axonemal heavy chain isoforms
Authors: Asai David J. , Beckwith Susan M. , Kandl Kimberly A. , Keating Heather H. , Tjandra Hendri , Forney James D.
Published: April 2021

Paramecium tetraurelia is a unicellular organism that utilizes both axonemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-directed polymerase chain reaction. Eight different P-loop-containing cDNA fragments were cloned. Southern hybridization analysis indicated that each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Seven of the eight cloned contain sequence motif A, which is found in axonemal dyneins, and one contains sequence motif B, which is found in the dyneins from cell types that do not have cilia or flagella. Two of the Paramecium dynein genes were further investigated: DHC-6 which contains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary β heavy chain and DHC-8 is similar to the cytoplasmic dynein from Dictyostelium. Deciliation of the cells resulted in a substantial increase in the steady state concentration of DHC-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Competitive solid-phase binding assays demonstrated that each antiserum was peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bovine brain but did not react with ciliary dynein. Indirect immunofluorescence microscopy showed that the motif B epitope was excluded from cilia. These results suggest that sequence motif A may be present in both classes of dynein but that sequence motif B is restricted to cytoplasmic dynein isoforms. This study provides direct evidence that individual dynein gene products are spatially segregated in a single cell.
Nuclear export of signal recognition particle RNA is a facilitated process that involves the Alu sequence domain
Authors: He Xiao-Ping , Bataillé Nelly , Fried Howard M.
Published: April 2021

The signal recognition particle is a cytoplasmic RNA-protein complex that mediates translocation of secretory polypeptides into the endoplasmic reticulum. We have used a Xenopus oocyte microinjection assay to determine how signal recognition particle (SRP) RNA is exported from the nucleus. Following nuclear injection, SRP RNA accumulated in the cytoplasm while cytoplasmically injected SRP RNA did not enter the nucleus. Cytoplasmic accumulation of SRP RNA was an apparently facilitated process dependent on limiting trans-acting factors, since nuclear export exhibited saturation kinetics and was completely blocked either at low temperature or by wheat germ agglutinin, a known inhibitor of nuclear pore-mediated transport. At least one target for trans-acting factors that promote nuclear export of SRP RNA appears to be the Alu element of the molecule, since a transcript consisting of only the Alu sequence was exported from the nucleus in a temperature-dependent manner and the Alu transcript competed in the nucleus for transport with intact SRP RNA. Although the identities of trans-acting factors responsible for SRP RNA transport are at present unknown, we suggest that proteins contained within the cytoplasmic form of SRP are candidates. Consistent with this idea were the effects of a mutation in SRP RNA that prevented binding of two known SRP proteins to the Alu sequence.