Showing papers in "Journal of Cell Science in 1989"
TL;DR: A set of monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof to define each structural component of this highly ordered cytos skeleton.
Abstract: The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.
651 citations
TL;DR: An assay for estimating the number of adherent cells present in a microculture and its application to the study of growth factors, which was shown to be applicable to a number of foetal and adult cell lines derived from man and experimental animals.
Abstract: There is currently much interest in the role of mediators that regulate cell proliferation. Methods to assay proliferative effects of such mediators usually involve cell counting techniques, which are tedious to perform, or methods based on uptake of radiolabelled thymidine, which may be prone to errors caused by precursor pool artefacts. We describe here an assay for estimating the number of adherent cells present in a microculture and its application to the study of growth factors. The assay depends on the binding of Methylene Blue to the fixed monolayer at pH 8.5 and, after washing the monolayer, release of dye by lowering pH. The use of an elution solvent containing acidified ethanol ensures a linear correlation between absorbance of the dye and cell number, and enables the assay to be carried out in 96-well plates measuring absorbance with an automated vertical light-path microplate photometer. The assay is rapid, highly reproducible and easy to perform, making it ideal for screening large numbers of samples. It was shown to be applicable to a number of foetal and adult cell lines derived from man and experimental animals. It was also demonstrated to be useful for assaying purified growth factors and detecting growth promoting activity in cell and tissue extracts.
435 citations
TL;DR: The silicone rubber substratum technique was used in combination with fluorescence microscopy in order to observe the effects of microtubule-depolymerizing drugs on the contractile strength and organization of cytoplasmic actin networks.
Abstract: Despite considerable evidence that cytoplasmic microtubules play some role in guiding or controlling the locomotion of tissue cells, the nature of this control is not understood. In particular, little is known about the role of microtubules in the exertion of propulsive 'traction' forces, or about microtubule effects on the organization of the cytoplasmic actin stress fibers. In this study, the silicone rubber substratum technique was used in combination with fluorescence microscopy in order to observe the effects of microtubule-depolymerizing drugs on the contractile strength and organization of cytoplasmic actin networks. Perfusion with a variety of microtubule poisons (either colcemid, nocodazole or vinblastine) was found to cause a rapid and substantial strengthening of fibroblast contractility. This was demonstrated in two established fibroblast cell lines, as well as in primary cultures of rat gingival fibroblasts and embryonic chick heart fibroblasts. Treatment with the drug taxol, which promotes microtubule assembly, was found to prevent the strengthening effects of the microtubule inhibitors. It was also found that the disruption of actin stress fibers by the phorbol ester tumor promoter, TPA, is reversed by microtubule poisons: stress fibers reform within 30 min of the addition of the microtubule drugs, despite the continued presence and activity of the TPA. Several possible mechanisms are considered, including the idea that microtubule assembly normally exerts a pushing force, which counterbalances part of the contractile force exerted by the actin stress fibers. However, the mechanism that seems best to account for the observations is that microtubules modulate, in an inhibitory fashion, the contractility and the state of organization of cytoplasmic actin.
315 citations
TL;DR: The invasive phenotype, which in its keratin profile corresponds to the differentiated luminal cell and that of the metastatic cancer lines, cannot be cultured from primary breast cancers using MX, which supports proliferation of the corresponding normal cell.
Abstract: The luminal and basal epithelial cells in the human mammary gland can be distinguished in tissue sections on the basis of the pattern of keratins they express. Moreover, the invasive cells in primary carcinomas show a keratin profile that corresponds to that of the dominant luminal cell (7, 8, 18, 19). When homogeneous populations of luminal epithelial cells from milk or from breast cancer metastases are cultured the profile of keratin expression seen in vivo is maintained. We have therefore used monospecific antibodies reactive with individual keratins to examine the phenotype of cells cultured in three different media from reduction mammoplasty tissue that contains both luminal and basal cells. The phenotype of cells cultured from primary breast cancers in one of these media (MCDB170) has also been examined. In characterizing cell phenotypes, antibodies to a polymorphic epithelial mucin (PEM) expressed in vivo by luminal cells, and to smooth muscle (a) actin, expressed in vivo by basal cells, have also been used. Our results show that proliferation of different cell phenotypes is selected for in different media. In milk mix (MX) developed for growth of luminal cells from milk, only the luminal cell phenotype proliferates (for only 1 or 2 passages). In medium MCDB 170, which was developed for long-term growth of human mammary epithelial cells from reduction mammoplasty organoids, cells from the basal layer proliferate, while in MM medium the basal phenotype dominates, but a few cells with the luminal phenotype are found. Around passage 3, in medium MCDB 170, most cells senesce and a subpopulation of cells proliferates on further passage. These cells retain expression of the basal epithelial keratins but also express some features characteristic of luminal epithelial cells, suggesting that the basal layer may contain a stem cell that can develop along the luminal lineage. In culture, however, they do not express keratin 19, which in vivo is a feature of the fully differentiated luminal cell. The cells cultured from primary breast cancer in medium MCDB 170 have a similar keratin profile to that of the normal cells cultured in this medium. They do not express keratin 19, even though the invasive cells in primary cancers homogeneously express this keratin in vivo. The invasive phenotype, which in its keratin profile corresponds to the differentiated luminal cell and that of the metastatic cancer lines, cannot be cultured from primary breast cancers using MX, which supports proliferation of the corresponding normal cell.
271 citations
TL;DR: Test directly the stability of acetylated MTs by determining their specific rate of turnover, which indicated that acetylation modified discrete regions along stable MTs can be extremely long-lived.
Abstract: Tubulin is subject to a post-translational acetylation reaction that is thought to be correlated with increased stability of the modified microtubules (MTs). We sought to test directly the stability of acetylated MTs by determining their specific rate of turnover. We used human fibroblasts, which contain a subset of MTs that display terminal and internal domains of acetylation. The turnover of acetylated domains was analysed by microinjecting cells with biotinylated brain tubulin and determining, by triple-label immunofluorescence, the progress of incorporation of biotinylated tubulin into acetylated and non-acetylated domains. Within two minutes after injection, biotinylated domains were contiguous with virtually all observed non-acetylated MT ends but were not contiguous with terminal acetylated domains, demonstrating that the former were growing while the latter were not. Ten minutes after injection, many MTs lacking acetylated domains had incorporated biotinylated subunits uniformly while most MTs containing acetylated domains remained unlabelled, indicating that non-acetylated MTs were turning over while most acetylated domains were not. One hour after injection, virtually all non-acetylated MTs were labelled with biotin whereas approximately half of the acetylated domains contained biotin, demonstrating that acetylated domains turned over much more slowly than the non-acetylated, bulk array. Non-acetylated MT regions flanking acetylated domains also lacked hapten, indicating that acetylation modified discrete regions along stable MTs. Sixteen hours after injection, cells that had not entered mitosis still retained acetylated domains that had not turned over (13% of all acetylated domains), indicating that acetylated domains can be extremely long-lived.(ABSTRACT TRUNCATED AT 250 WORDS)
265 citations
TL;DR: It is suggested that macropinocytosis increases the size difference between pinosomes and efflux vesicles, and that that difference increases greatly both solute accumulation and membrane flow through the endocytic compartment.
Abstract: The morphology and kinetics of pinocytosis by bone marrow-derived macrophages were studied to determine how stimulation by phorbol esters increases net solute accumulation. Application of phorbol myristate acetate (PMA) increased both the abundance of macropinosomes and the rate of solute flow through the endocytic compartment. The large pinosomes originated as ruffles at the cell margins that folded back on themselves, internalizing extracellular medium and solutes. I examined how stimulation affects the kinetics of pinocytic influx, accumulation, and subsequent efflux of the fluorescent dye Lucifer Yellow (LY) in macrophages. Both the accumulation of LY and its subsequent efflux were temperature-dependent and directly proportional to the concentration of LY in the extracellular medium. Macrophages incubated in PMA and LY for 2 h accumulated four to six times more LY than did macrophages in LY alone. If after pinocytosis the macrophages were washed and reincubated in unlabeled medium for a 1 h chase period, some of the internalized LY was regurgitated from the cells. Inclusion of PMA in the chase medium increased efflux of LY. In contrast, a smaller percentage of LY was regurgitated from macrophages which were both loaded and chased in the presence of PMA. This indicates that although efflux is increased by PMA, influx increases more, and therefore more of the LY entering by pinocytosis is retained within the cell. I suggest that macropinocytosis increases the size difference between pinosomes and efflux vesicles, and that that difference increases greatly both solute accumulation and membrane flow through the endocytic compartment.
247 citations
TL;DR: The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells and when mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold.
Abstract: In interphase cells the proliferation-associated antigen recognized by monoclonal antibody Ki-67 is almost exclusively located in the nucleoli. When cells at several stages of mitosis were examined for the localization of the Ki-67 antigen, a striking redistribution could be observed. During prophase the distinct nucleolar Ki-67 fluorescence changed to a bright irregular meshwork throughout the nucleoplasm. At metaphase the antigen appeared to be distributed in a reticulate structure surrounding the condensed chromosomes, while at late telophase a punctated staining of the entire nucleoplasm was observed, which preceded the typical nucleolar localization pattern in each of the two daughter cells. Immunolabelling with Ki-67 of metaphase chromosome spreads revealed a circumferential staining of the individual chromosomes. The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells. When mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold. Quantification of the Ki-67 fluorescence signal using flow cytometry revealed the highest staining intensities in mitotic cells. Furthermore, it was shown that nutritionally deprived cells became negative for Ki-67.
240 citations
TL;DR: Testing of cell cultures of different species by means of indirect immunofluorescence revealed that the Ki-67 antibody reacted with human cells and with the Rhesus monkey kidney-derived cell line LLC-MK2, and the antigen was not identified in immunoblotting or immunoprecipitation assays.
Abstract: Ki-67 is a commercially available mouse monoclonal antibody, which reacts with a nuclear antigen in proliferating cells. The antibody can be used to determine the growth fraction of human tumours in situ and has been shown to be of prognostic importance. In this study it is shown that in interphase cells Ki-67 reacts with an antigen, mainly present in the nucleoli. Confocal scanning laser microscopy and immunoelectron microscopy on human MR65 monolayer cells revealed that this nucleolar antigen is predominantly localized in the nucleolar cortex and in the dense fibrillar components. The Ki-67 antigen appeared to be preserved in nuclear matrix preparations obtained after in situ fractionation of MR65 cells. Despite many efforts, we could not identify the antigen in immunoblotting or immunoprecipitation assays. Testing of cell cultures of different species by means of indirect immunofluorescence revealed that the antibody reacted with human cells and with the Rhesus monkey kidney-derived cell line LLC-MK2.
213 citations
TL;DR: Although dependent upon vitronECTin for adhesion to the substratum, bovine endothelial cells were unable to synthesize endogenous vitronectin, which was shown to be due to a failure of fibronctin to coat the substrata in the presence of other serum proteins.
Abstract: The effects of vitronectin and fibronectin upon the attachment and growth of bovine corneal endothelial cells (BCE) and BHK-21 cells were compared. Similar dose-response curves for cell attachment to the substratum were obtained for both molecules and both cell types, although BCE cells exhibited a slight preference for vitronectin, and BHK cells for fibronectin. When, however, cells were plated in medium containing bovine serum stripped of fibronectin, they attached and grew normally, whereas in medium containing serum stripped of vitronectin, cells either failed to attach (BHK-21) or attached but exhibited poor cell spreading and growth. This dependence of cells upon vitronectin, rather than fibronectin, in serum for cell attachment, was shown to be due to a failure of fibronectin to coat the substratum in the presence of other serum proteins. Vitronectin was able to coat the substratum efficiently in the presence of other serum proteins. Although dependent upon vitronectin for adhesion to the substratum, bovine endothelial cells were unable to synthesize endogenous vitronectin.
213 citations
TL;DR: Analysis of nuclei fixed early in replication reveals that these foci of DNA replication number about 100-300 for each nucleus and probably represent the replicon clusters already described for tissue culture cells, indicating that replication forks remain tightly clustered in groups of at least 300 throughout the period of replication.
Abstract: Demembranated Xenopus sperm nuclei were induced to replicate synchronously in a low-speed supernatant (LSS) of Xenopus eggs by preincubation in a high-speed supernatant (HSS). DNA replication was observed by incorporation of [alpha-32P]dATP, BrdUTP or biotin-dUTP. Biotin-dUTP incorporation, visualised with fluorescent streptavidin, reveals a striking pattern of replication foci throughout replicating nuclei. We show that this represents a precursor to the bright uniform fluorescence seen later. Confocal microscopic analysis of nuclei fixed early in replication reveals that these foci of DNA replication number about 100–300 for each nucleus and probably represent the replicon clusters already described for tissue culture cells. Foci are evenly distributed throughout the nuclei and are not concentrated at or near the nuclear envelope. Complete replication of each nucleus occurs in an average time of only one hour in this system. Hence we calculate that there must be at least 300–1000 replication forks together in each cluster. Furthermore, pulse labelling at later times in the period of replication reveals a similar pattern of foci indicating that replication forks remain tightly clustered in groups of at least 300 throughout the period of DNA replication.
187 citations
TL;DR: The invariant handedness of the spindle pole, eyespot position, and mating structure position appears to be based on the inherent asymmetry of the basal body pair, providing an example of how an intracellular pattern can be determined and maintained.
Abstract: Although largely bilaterally symmetric, the two sides of the unicellular alga Chlamydomonas reinhardtii can be distinguished by the location of the single eyespot. When viewed from the anterior end, the eyespot is always closer to one flagellum than the other, and located at an angle of approximately 45 degrees clockwise of the flagellar plane. This location correlates with the position of one of four acetylated microtubule bundles connected to the flagellar apparatus. Each basal body is attached to two of these microtubule rootlets. The rootlet that positions the eyespot is always attached to the same basal body, which is the daughter of the parental/daughter basal body pair. At mitosis, the replicated basal body pairs segregate in a precise orientation that maintains the asymmetry of the cell and results in mitotic poles that have an invariant handedness. The fusion of gametic cells during mating is also asymmetric. As a result of asymmetric, but different, locations of the plus and minus mating structures, mating preferentially results in quadriflagellate dikaryons with parallel flagellar pairs and both eyespots on the same side of the cell. This asymmetric fusion, as well as all the other asymmetries described, may be necessary for the proper phototactic behavior of these cells. The invariant handedness of the spindle pole, eyespot position, and mating structure position appears to be based on the inherent asymmetry of the basal body pair, providing an example of how an intracellular pattern can be determined and maintained.
TL;DR: Immolocalization of keratin 17 provides evidence that the expression of this keratin strongly depends on the cell position within epithelial structures, and suggests that these proteins may be implicated in the generation of spatial organization of epithelial tissues.
Abstract: By immunomorphology, using keratin 17-specific monoclonal antibody, it has been shown that this keratin is expressed only in the basal cells of a group of complex epithelia: glandular epithelium with myoepithelial component, transitional and pseudostratified epithelia. Immunolocalization of keratin 17 provides evidence that the expression of this keratin strongly depends on the cell position within epithelial structures. The topographical character of the keratin expression suggests that these proteins may be implicated in the generation of spatial organization of epithelial tissues.
TL;DR: It was found that during normal aggregation oscillation frequency increases while at the same time wave propagation velocity decreases, which may indicate a more vigorous chemotactic response by individual cells or a better synchronization of the responding cell populations due to shortened Chemotactic deadaptation times.
Abstract: Waves of chemotactic movement during the early phase of aggregation in Dictyostelium discoideum were analyzed by digital image processing in a manner that immediately shows the following parameters: wave propagation velocity, period length, wave amplitude und wave shape. We have characterized the aggregation of AX-2 and the streamer F mutant NP 377 in terms of these parameters and investigated the influence of caffeine and ammonia. It was found that during normal aggregation oscillation frequency increases while at the same time wave propagation velocity decreases. Caffeine, a known inhibitor of cyclic AMP relay, reduces oscillation frequency and wave propagation velocity in a dose-dependent manner but most notably leads to the appearance of bimodal (harmonic) oscillations. These bimodal waves are also found in streamer F mutants without caffeine during early aggregation. The effect of caffeine is interpreted as an increase in the average chemotactic deadaptation time due to elevated cyclic GMP levels after a cyclic AMP stimulus. This increased deadaptation time results in some cells responding to every chemotactic signal, while others respond only to every second signal, leading to mixed population behavior and hence biphasic optical density waves. Ammonia has no significant influence on oscillation frequency and wave propagation velocity but shows a clear increase in the amplitude of the optical density waves. This may indicate a more vigorous chemotactic response by individual cells or a better synchronization of the responding cell populations due to shortened chemotactic deadaptation times.
TL;DR: A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies and both anti-S-1 and anti-LMM stains both the generative cell and the vegetative nuclear envelope.
Abstract: A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies. The epitopes of these antibodies were found to reside on the myosin heavy chain head and rod portion and were, therefore, designated anti-S-1 (myosin S-1) and anti-LMM (light meromyosin). On Western blots of the total soluble pollen tube proteins, both anti-S-1 and anti-LMM label a polypeptide of approximately 175,000 Mr. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the whole length of the tube, often with an enrichment in the tube tip. These fluorescent spots are thought to represent vesicles and/or organelles in the pollen tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope. The different staining patterns of the nucleus between anti-S-1 and anti-LMM may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and/or organelles.
TL;DR: This paper reviews the current knowledge of the cyclins based on observations of the oocytes and eggs of sea urchins, clams and frogs, and describes various conditions under which cyclin destruction is delayed or deranged.
Abstract: Summary This paper reviews our current knowledge of the cyclins based on observations of the oocytes and eggs of sea urchins, clams and frogs. Cyclins are proteins found in all eukaryotes whose special property is rapid destruction at specific stages in the cell cycle. The cyclins fall into three families. A-type cyclins have been found in clams, flies and frogs. B-type cyclins have been found in clams, flies, frogs, sea urchins and fission yeast. A more distantly related family of three genes is found in Saccharomyces cerevisiae. B-type cyclins appear to be required for cells to enter mitosis, and their destruction is thought to be necessary for exit from mitosis. We describe evidence in support of these ideas, and describe various conditions under which cyclin destruction is delayed or deranged. We conclude with a discussion of the relationship between the cyclins and maturation- (or M phase-) promoting factor and some ideas on how the cyclins may work.
TL;DR: This finding suggests that an actomyosin motility system is present in pollen tubes, and indicates that the movements of the different classes of inclusions are driven by interaction of the surface myosin with the actin fibrils at the zones of contact.
Abstract: Myosin, detected by immunofluorescence using an antibody to bovine skeletal and smooth muscle myosin, has been localised on individual identifiable organelles from the grasses Alopecurus pratensis and Secale cereale, and on the surfaces of vegetative nuclei and generative cells from pollen and pollen tubes of Hyacinthus orientalis and Helleborus foetidus. Taken in conjunction with recent evidence showing that the growing pollen tube contains an actin cytoskeleton consisting of numerous mainly longitudinally oriented microfilament bundles, and that isolated pollen-tube organelles show ATP-dependent movement along the actin bundles of the giant cells of the characeous algae, this finding suggests that an actomyosin motility system is present in pollen tubes, and indicates that the movements of the different classes of inclusions are driven by interaction of the surface myosin with the actin fibrils at the zones of contact.
TL;DR: In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction.
Abstract: Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108,000 (cingulin-108), with a Stokes' radius of approximately 15 nm, and a molecular length of 130 nm +/- 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. In permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vinculin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (zonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.
TL;DR: The experiments reported here characterise further the requirements for the induction of cytotoxicity by polycations and find that the extent of inhibition is related to both the identity of the monomer and the configuration of the polymers.
Abstract: Pro-inflammatory effects of cationic proteins secreted by human granulocytes include induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. We have shown previously that a series of synthetic polycationic amino acids produce a dose-, time- and Mr-dependent inhibition of [3H]leucine or [3H]thymidine incorporation into macromolecules by human umbilical vein endothelial cells, and that the extent of inhibition was correlated with changes in cell morphology, with release of cytoplasmic constituents and was irreversible. The experiments reported here characterise further the requirements for the induction of cytotoxicity by polycations. We have found that the extent of inhibition is related to both the identity of the monomer, for polymers of Mr 40,000 the order is ornithine greater than lysine greater than arginine, and to its configuration; poly-D-lysines are more potent inhibitors than poly-L-lysines of similar Mr. Only brief exposure to the agonist is required, 90% inhibition occurred after 10 min of exposure to poly-L-lysine (Mr 90,000). Treatment of endothelial cells with neuraminidase, heparinase, hyaluronidase, chondroitinase or trypsin did not reduce their susceptibility to polylysine. Inhibition of microtubule or microfilament formation also had no effect on polylysine cytotoxicity, indicating that internalisation of the polymer was not a prerequisite for the effect. Inhibition of protein synthesis or pretreatment with simple sugars likewise failed to block the effects of polylysine treatment. Natural cationic proteins exerted similar effects on endothelial cells, the extent of the effect apparently being related to the pI of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: It is suggested that mf-associated structures such as filasomes constitute dense knots of actin network that function in localized cell wall growth by controlling the deposition of cytoplasmic vesicles.
Abstract: Changes in the ultrastructure of the fission yeast Schizosaccharomyces pombe during the cell division cycle were analyzed by three-dimensional reconstruction of serial section electron micrographs of freeze-substituted cells. Cytoplasmic vesicles were found at the cell ends during interphase and at the equatorial zone of cells undergoing cytokinesis. Filasomes behaved in a similar but temporally retarded way to vesicles. Microfilament(mf)-associated granules were found attached to the plasma membrane at the growing ends. Microfilaments were identified against the plasma membrane and adjacent to developing septa. From these observations it is suggested that mf-associated structures such as filasomes constitute dense knots of actin network that function in localized cell wall growth by controlling the deposition of cytoplasmic vesicles. Dictyosomes occur as tubular and fenestrated cisternae with associated cytoplasmic vesicles. They were distributed uniformly in the cytoplasm and did not change significantly during the cell cycle. Changes in the three-dimensional localization of cytoplasmic microtubules and mitochondria are also described.
TL;DR: Oligodendrocytes are inhibitory to axonal growth, and this may partially explain the failure of axons to regenerate in the mammalian central nervous system.
Abstract: We have examined the interactions between axons regenerating from dorsal root ganglia (DRGs) derived from newborn rats and oligodendrocytes cultured by three different techniques. Cultures examined after 2 days have a profuse outgrowth of axons from the DRGs, forming a dense mat on the culture surface. However, the axons avoid growing on oligodendrocytes; axons are seen all around these cells, but do not grow over them. We have also performed time-lapse video studies of the interactions between axonal growth cones and oligodendrocytes. Axons grow normally until their growth cone comes into direct contact with an oligodendrocyte, following which the growth cone remains motile for 30-60 min, but without making any progress over the cell. The growth cone then suddenly collapses, and the axon retracts, leaving a thin strand in contact with the cell. After this a new growth cone is usually elaborated and the process repeated. Oligodendrocytes are therefore inhibitory to axonal growth, and this may partially explain the failure of axons to regenerate in the mammalian central nervous system.
TL;DR: The data suggest that the synthesis of relatively high levels of HA by foetal fibroblasts at confluence may be causally related to the elevated migration displayed by these cells, and that there is a considerable degree of heterogeneity amongst the nine different fibroblast lines examined in this study in terms of the size class of HA.
Abstract: We have previously reported that confluent foetal fibroblasts migrate into three-dimensional collagen gel matrices to a significantly greater extent than do adult cells. Hyaluronic acid (HA) is a major constituent of the extracellular matrix deposited by fibroblasts and has been demonstrated to stimulate the migration of a number of different cell types. Previous studies have indicated that the synthesis of HA by normal adult skin fibroblasts declines significantly when the cells achieve confluence. Data presented in this paper indicate that foetal fibroblasts differ from adult cells in this respect, in that they do not show an inverse relationship between cell density and HA synthesis, i.e. confluent foetal fibroblasts continue to produce approximately the same amount of HA as do subconfluent cells. These data suggest that the synthesis of relatively high levels of HA by foetal fibroblasts at confluence may be causally related to the elevated migration displayed by these cells. In this context, a close correlation was observed between the level of HA synthesized by confluent foetal and adult fibroblasts and the differential migratory activity displayed by these cells. Such differences in HA synthesis and migratory behaviour were only apparent at cell confluence, with subconfluent foetal and adult fibroblasts being indistinguishable in terms of these two criteria. Our data further reveal that: (1) cell density affects the size class of HA synthesized by both foetal and adult cells; and that (2) there is a considerable degree of heterogeneity amongst the nine different fibroblast lines examined in this study in terms of the size class of HA that they produce.
TL;DR: It is proposed that the cell does not discriminate between these different integrins when assembling the cytoskeletal components at the cytoplasmic face of focal contacts as well as the vitronectin receptor under normal culture conditions.
Abstract: The distribution of two integrins, the fibronectin receptor and the vitronectin receptor, has been explored in an endothelium-derived cell line plated onto various substrata. On a fibronectin substratum, in the presence of serum, these cells develop focal contacts that contain the fibronectin receptor, whereas the vitronectin receptor is diffusely distributed over the cell surface. Conversely, cells plated onto vitronectin-coated coverslips concentrate only the vitronectin receptor within focal contacts. The accumulation of the vitronectin receptor within focal contacts also occurs when the cells are plated on uncoated coverslips but in the presence of serum. Therefore, we conclude that under normal culture conditions (i.e. in serum-containing media), the vitronectin receptor is the predominant form of integrin involved in substratum adhesion. This conclusion is supported by experiments in which cells were cultured on fibronectin-coated coverslips in the presence of serum. Initially these cells developed focal contacts containing only the fibronectin receptor. Within several hours, however, there was a progressive replacement of focal contacts containing the fibronectin receptor by focal contacts expressing the vitronectin receptor. After approximately 12 h in culture, most cells contained focal contacts expressing only the vitronectin receptor. Focal contacts containing either the fibronectin or vitronectin receptor were both associated with the termini of stress fibres and contained the proteins talin and vinculin. These observations lead us to propose that the cell does not discriminate between these different integrins when assembling the cytoskeletal components at the cytoplasmic face of focal contacts.
TL;DR: The results suggest a complete structural homology between axonemal and cytoplasmic dynein, and a model for the structural relationship between the two molecular forms is presented.
Abstract: Cytoplasmic dynein was purified from pig brain, using a modified version of published procedures, in order to study its interaction with microtubules. Since the preparation produces ATP-dependent sliding of taxol-stabilized purified microtubules over glass and runs on SDS-containing gels as a major band exceeding 300,000 Mr plus a medium chain band at about 75,000 Mr, it is assumed to be identical to the mammalian brain dynein (MAP 1C) purified by Vallee and colleagues. When viewed by electron microscopy in negative stain, individual particles show two distinct configurations. Some are clearly similar to the two-headed bouquet structure already shown for MAP 1C. A larger number of molecules in the present preparation appear to have two heads fused together, forming a dimeric globular particle with two separate tails. They are referred to as phiparticles, because of their resemblance to the greek letter phi. A model for the structural relationship between the two molecular forms is presented. The stems of two associated dynein subunits may separate beyond the base, to form a bouquet, or they may remain fused to form the larger tail of a phi-particle. The smaller tail probably represents a combined pair of features equivalent to the 'stalks' shown to emanate from axonemal dynein heads by Goodenough and colleagues. Both tails of a phi-particle can bind to microtubules, even in the presence of ATP, and cause microtubule bundling. These results suggest a complete structural homology between axonemal and cytoplasmic dynein.
TL;DR: Investigation of the transition from the rapid, early cell cycle to the slower, more somatic-like cell cycle that occurs after division twelve in developing Xenopus embryos, a stage called the mid-blastula transition (MBT), has shown that at high concentrations of nuclei the in vitro cycle is extended.
Abstract: The rapid, early cell divisions in Xenopus laevis embryos are driven by an inflexible oscillator that is not influenced by the state of the DNA. In contrast, mitosis in somatic cells can be prevented by blocking replication or by damaging the DNA through irradiation. We have investigated the transition from the rapid, early cell cycle to the slower, more somatic-like cell cycle that occurs after division twelve in developing Xenopus embryos, a stage called the mid-blastula transition (MBT). When aphidicolin, an inhibitor of DNA synthesis, was added to embryos just post-fertilization, the embryos continued to divide despite incomplete replication. Also, embryos incubated with aphidicolin from early times did not slow their cell cycles after division twelve as control embryos did, indicating a connection between the accumulation of DNA and the post-MBT timing of the cell cycle. However, incubation with hydroxyurea, an inhibitor of ribonucleotide reductase, resulted in an S phase arrest when the pools of dNTPs became depleted after division twelve. These experiments showed that the embryos had acquired the ability to arrest in S phase some time after the early divisions and before division thirteen. The acquisition of the ability to arrest in S phase did not depend upon new transcription. These experiments suggested that the number of nuclei present could be responsible for the extension of the cell cycle observed after the MBT. To investigate this, we added increasing concentrations of nuclei to an in vitro cell cycle system. We have shown that at high concentrations of nuclei the in vitro cycle is extended.
TL;DR: It is proposed that the haemolysin is translocated directly to the medium bypassing the periplasm and that HlyB and HlyD together constitute a membrane-bound translocator specific for molecules bearing the HlyA targeting sequence, and that the organization of this complex must somehow straddle the inner and outer membranes.
Abstract: Escherichia coli haemolysin (HlyA), a 107K (K = 10(3) Mr) protein, is secreted to the medium in an hlyB, hlyD-dependent process. Secretion, however, depends on neither an N-terminal signal sequence nor on SecA, which is part of the normal cellular export machinery for periplasmic and outer membrane proteins. In contrast, HlyA contains a novel C-terminal secretion signal encompassing the last 27 amino acids and possibly some additional residues immediately upstream. This region is characterized by a 16 residue 'aspartic acid box' composed largely of small amino acids which we propose constitutes an important element in recognition of the membrane translocation complex constituted by HlyB and HlyD. This feature is also found at the C-terminus of the adenyl cyclase and leukotoxin A molecules and resembles a recently identified eukaryotic C-terminal signal for targeting to glycosomes. A domain of the HlyB component of the haemolysin transport system is also similar to a domain widely distributed in nature, apparently acting as an ATP-dependent transport protein for a wide variety of molecules. Secretion of haemolysin, however, is the first example of a protein translocation system involving an HlyB-like molecule. This suggests that a major role of HlyB or at least its C-terminal domain is the coupling of energy to translocation of the haemolysin. It is more likely therefore that HlyD is more involved in the actual translocation through the membrane. On the basis of genetical and biochemical studies we propose that the haemolysin is translocated directly to the medium bypassing the periplasm. We further propose that HlyB and HlyD together constitute a membrane-bound translocator specific for molecules bearing the HlyA targeting sequence, and that the organization of this complex (conceivably involving other E. coli membrane proteins) must somehow straddle the inner and outer membranes. Finally, the HlyA C-terminal domain has been successfully used to promote the secretion to the medium of a number of heterologous polypeptides, in an HlyB,D-dependent manner.
TL;DR: The titin epitopes described here enable us to begin to correlate known ultrastructural aspects of the interior part of the A band with the disposition of the titin molecule in the sarcomere, and raise the question of whether there is a regular interaction pattern between titin and the thick filaments.
Abstract: A direct titin-thick filament interaction in certain regions of the A band is suggested by results using four new monoclonal antibodies specific for titin in immunoelectron microscopy. Antibodies T30, T31 and T32 identify quasi-repeats in the titin molecule characterized by a 42–43 nm repeat spacing. These stripes seem to coincide with striations established by others on negatively stained cryosections of the A band. Antibodies T30 and T32 recognize epitopes matching five or two of the seven striations per half sacromere known to harbor both the myosin-associated C-protein and an 86K (K = 10(3) Mr) protein. Antibody T31 labels two stripes in the P zone, which correspond to the two positions where decoration is seen with 86K protein, but not with C-protein. The single titin epitope defined by antibody T33 is located 55 nm prior to the center of the M band. This position seems to coincide with the M7 striation defined by others on negatively stained A bands. The T33 epitope position proves that the titin molecule, which is known to be anchored at the Z line, also penetrates into the complex architecture of the M band. The titin epitopes described here enable us to begin to correlate known ultrastructural aspects of the interior part of the A band with the disposition of the titin molecule in the sarcomere. They raise the question of whether there is a regular interaction pattern between titin and the thick filaments.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal Article•
TL;DR: It is suggested that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryogenesis in Drosophila.
Abstract: Using monoclonal antibodies, we followed the fate of three different nuclear envelope proteins during mitosis in Drosophila early embryos by indirect immunofluorescence microscopy. Two of these proteins, lamin and otefin, a newly characterized nuclear envelope polypeptide with an apparent Mr of 53,000, are apparently present in an envelope-like structure that is present throughout mitosis. Immunoelectron microscopy of interphase nuclei indicates that otefin, like lamin, is not a component of nuclear pore complexes. In contrast with lamin and otefin, gp188, a putative pore complex component, was completely redistributed through the surrounding cytoplasm during prophase in comparable early embryo specimens and was present in an envelope only in interphase. Together with previous morphological studies by other workers, these data suggest that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryogenesis in Drosophila. This ‘spindle envelope’, as it has been named by others, contains both lamin and otefin but probably not pore complex proteins.
TL;DR: The presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.
Abstract: In this paper we provide evidence that ectoplasmic specializations are a form of intercellular adhesion junction. Ectoplasmic specializations, found at basal junctions between adjacent Sertoli cells and at sites of adhesion between Sertoli cells and germ cells, consist of actin filament bundles sandwiched between the plasma membrane and a cistern of endoplasmic reticulum. The actin filaments in each bundle are unipolar and are hexagonally packed. The bundles are coupled to the adjacent membranes and to each other. Because ectoplasmic specializations are associated with junctional sites, they may play a role in intercellular adhesion. In this study, we report a procedure for obtaining samples enriched for ectoplasmic specializations and identify polypeptides that may be associated with ectoplasmic specializations. On SDS-polyacrylamide gels, an 83K (K = 10(3) Mr) polypeptide is specific to the ectoplasmic specialization-enriched sample, suggesting that it may be a component of ectoplasmic specializations. Other polypeptides at 38, 53, 56 and 69K also may be associated with ectoplasmic specializations. Immunoblots further indicate that fimbrin and vinculin are present in the ectoplasmic specialization-enriched fraction. In addition, immunofluorescence indicates that vinculin is associated with spermatid-Sertoli cell and Sertoli-Sertoli cell junctions. We suspect that fimbrin, an actin-bundling protein, may be involved in cross-linking the hexagonally packed actin filaments in ectoplasmic specializations while vinculin may be associated with actin-membrane linkages. If so, ectoplasmic specializations may be a new class of actin-associated junctional site. Moreover, the presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.
TL;DR: It is proposed that the formation of vimentin cables involves a phosphorylation event, while the coiling of cables into a perinuclear mass relies on interaction of intermediate filaments with a component of the actin cortex.
Abstract: In this study, we have investigated the properties of intermediate filament rearrangements using experimentally induced collapse of vimentin intermediate filaments in mouse fibroblasts. In these cells, depolymerizing microtubules by colchicine or vinblastine treatment at 37 degrees C results in a two-stage collapse of intermediate filaments. First, the vimentin filaments aggregate into large cables; then, the cables coil into a dense mass surrounding the nucleus. By using inhibitors of oxidative phosphorylation along with glucose deprivation to lower intracellular ATP levels by 95%, we have found that both stages of intermediate filament collapse require ATP. However, once collapse has occurred, only the second stage can be reversed in the absence of microtubules by lowering ATP levels. An additional difference between the two stages of collapse was revealed by treating cells with cytochalasin D: the formation of intermediate filament cables still occurs after disruption of the actin filament system by cytochalasin, but the subsequent coiling of cables to form a perinuclear mass is strongly inhibited by these conditions, and can be reversed by applying cytochalasin to cells in which intermediate filaments have already undergone complete collapse. We propose that the formation of vimentin cables involves a phosphorylation event, while the coiling of cables into a perinuclear mass relies on interaction of intermediate filaments with a component of the actin cortex.