bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence against a direct role for cortical actin arrays in saltatory organelle motility in hyphae of the fungus Saprolegnia ferax
Authors: Heath I. Brent
Published: April 2021

Actin stained by rhodamine-labelled phalloidin in growing hyphae of the oomycete fungus Saprolegnia ferax is restricted to an approximately 0–25 μm deep layer of the cell periphery where it forms an apical cap of fine filaments and a subapical array of coarser longitudinal fibrils interspersed with plaques. The functions of this actin are unknown, but because actin-rich fibrils in other cells are involved in organelle motility I have sought evidence for a similar role in these hyphae. The most prominent motile structures are a population of spherical, predominantly sub-micrometre diameter, refractile vesicles of unknown function, which show typical saltatory movements along the hyphae. The motility of these vesicles is statistically identical in both the central cytoplasm, remotefrom the actin fibrils, and the peripheral cytoplasm adjacent to the fibrils. Treatment of hyphae with specific concentrations and durations of the detergents Tween 20 and Brij 58 causes extensive reorganization of the actin arrays with no effect on vesicle motility, whereas Triton X-100 severely inhibits motility with little detectable effect on the actin filaments. These observations show that normal vesicle motility does not depend on the proximity of a normal population of actin filaments and, therefore, suggest that the latter have some function other than a direct role in saltatory organelle motility.
Electron spectroscopic imaging of the centrosome in cells of the indian muntjac
Authors: Rattner J. B. , Bazett-Jones D. P.
Published: April 2021

Specific antibody labelling indicates that phosphoproteins are present at microtubule-organizing centres, including the centrosome. We have employed electron spectroscopic imaging techniques that permit high-resolution elemental analysis of thin sections of intact cells to investigate the precise distribution of phosphorus and therefore phosphoproteins at the centrosome of Indian muntjac cells. We report that these proteins are localized to both the pericentriolar matrix and the centriole. The matrix contains an abundance of phosphorus and is associated with microtubule elements.

Within the mature centriole, major structures including the nine triplet blades and linking elements that connect adjacent blades are composed of phosphorylated proteins. In addition, phosphoproteins are abundant at the ends of the centriole, at the interface between the centriole lumen and the pericentriolar environment. From these observations we suggest that phosphoproteins may play both a structural and a functional role within the centrosome region.
Dynamic changes in viscoelastic properties in cytotoxic T-lymphocyte-mediated killing
Authors: Sung Kuo-Li P. , Sung Lanping A. , Crimmins Mary , Burakoff Steven J. , Chien Shu
Published: April 2021

The biophysical properties of cytotoxic T lymphocytes during the killing of their target cells was investigated by using a human cytotoxic T lymphocyte clone, Fl, and the target cell, JY, for which it is specific. In single cytotoxic cell/target cell pairs after their conjugation there are changes in the viscoelastic properties of the target cell in association with the lethal hit delivery and post-binding cytolytic steps. On the basis of these changes in the target cell, the complex cytolytic event can be divided into stages: the viscoelastic coefficients exhibited an initial increase followed by a return to resting values; thereafter these coefficients decreased below control and then rose again prior to lysis. The eventual killing of the target cell involves bubbling and swelling of the nucleus, clustering of granules, damage to the cytoplasmic membrane, cell swelling, and lysis. The viscoelastic changes involved in target cell death suggest the loss of integrity of its cytoskeletal apparatus.
The extracellular matrix of hybrids between melanoma cells and normal fibroblasts
Authors: Copeman Michael C. , Harris Henry
Published: April 2021

It has been shown that when malignant tumour cells are fused with normal fibroblasts the suppression of malignancy in the hybrids is linked to their ability to produce a collagenous extracellular matrix in vivo. When, as a consequence of chromosome loss, segregants arise that reacquire malignancy, these do not produce any detectable matrix. In this paper we examine the main components of the extracellular matrix produced in vitro by hybrids between malignant mouse melanoma cells and normal mouse fibroblasts. Hybrids in which malignancy is suppressed synthesize about ten times as much type 1 procollagen as the malignant segregants derived from them; they also retain more fibronectin in the cell layer and release less protease activity into the medium. Malignant segregants more closely resemble the parental melanoma cells in producing fibronectin and mainly types IV and V procollagen. When hybrid cells in which malignancy is initially suppressed are grown continuously in vitro, the production of type I procollagen declines, and the production of type V procollagen and the release of protease activity into the medium increase. These changes, which are associated with the loss from the hybrid cells of both copies of the chromosome 4 derived from the parental fibroblast, predict the reacquisition of malignancy when the cells are inoculated into mice. It is possible that one gene or set of genes located on chromosome 4 determines both the execution of the fibroblast differentiation programme and the suppression of malignancy.
Non-specific elongation of cell cycle phases by cycloheximide in rat 3Y1 cells, and specific reduction of G1 phase elongation by simian virus 40 large T antigen
Authors: Okuda Atsuyuki , Kimura Genki
Published: April 2021

Partial inhibition of protein synthesis by cycloheximide caused prolongation of Gi, S and G2 phases in rat 3Y1 fibroblasts. In cells expressing simian virus 40 (SV40) large T antigen, by infection with SV40 in the previous generation, the prolongation of G1phase in the presence of cycloheximide was suppressed. However, the prolongation of S and G2 phases in the presence of cycloheximide was not suppressed in cells expressing large T antigen, by infection with SV40 in the current generation. Similarly, when density-arrested cells (cells in G0 phase) were infected with SV40 (either wild-type strain or a mutant deleted in the unique coding region for small t antigen) and reseeded sparsely in the presence of cycloheximide, the cycloheximide-induced delay of entry into S phase was suppressed. In this case, the reduction in [35S]methionine incorporation, that in protein accumulation and that in cell volume increase, were not surmounted by SV40 infection. In T-antigen-negative cells, all the regions in G1 phase seemed to be sensitive to cycloheximide, i.e. they suffered elongation. These results suggest that, in comparison with cells that enter S phase by the action of growth factors, cells expressing large T antigen can enter S phase more efficiently through a quite different process.
Interaction of mouse sperm with purified sperm receptors covalently linked to silica beads
Authors: Vazquez Monica H. , Phillips David M. , Wassarman Paul M.
Published: April 2021

We describe a solid-phase assay that bas permitted further analysis of zona pellucida glycoprotein, ZP3, as sperm receptor and acrosome reaction-inducer during fertilization in mice. The assay employs silica beads that contain epoxy groups to which purified, mouse oocyte ZP3 is covalently linked (ZP3-beads). ZP3-beads were characterized, e.g. by whole-mount autoradiography and flow cytofluorometry, incubated with capacitated mouse sperm under a variety of conditions, and the extent of sperm binding determined by light microscopy. Results of experimenta presented suggest the fol-lowing: (1) sperm bind specifically to ZP3-beads, but not to silica beads either exposed to 2-aminoeth-anol or derivatized with oocyte ZP2, fetuin or bovine serum albumin. (2) In nearly ail cases, only one sperm binds per ZP3-bead and binding occurs via the sperm head. (3) The extent of sperm binding to ZP3-beads is dependent on ZP3 and sperm concentrations, as well as on incubation time and temperature. (4) Sperm binding to ZP3-beads is unaffected by antibodies directed against ZP3, but is inhibited in a reversible manner by treatment of ZP3-beads with galactose oxidase. (5) Only acro-some-intact sperm bind to ZP3-beads but, once bound, sperm can undergo the acrosome reaction, which results in their release from ZP3-beads. (6) Islet-activating protein and 3-quinuclidinyl ben-zilate, two inhibitors of the zona pellucida-induced acrosome reaction, prevent sperm bound to ZP3-beads from undergoing the acrosome reaction. These results confirm and extend previous studies of sperm-egg interaction in mice, and suggest that the solid-phase assay will be useful for both cellular and biochemical analyses of mammalian fertilization.
Isolation and biochemical characterization of septate junctions: Differences between the proteins in smooth and pleated varieties
Authors: Lane Nancy J. , Dilworth Stephen M.
Published: April 2021

Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22 000–24 000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.
Monoclonal antibody against the centrosome
Authors: Rao Potu N. , Zhao Ji-Ying , Ganju Ramesh K. , Ashorn Cheryl L.
Published: April 2021

A monoclonal antibody, MPM-13, raised against mitotic HeLa cell extracts recognized a perinuclear area in interphase cells and spindle poles in mitotic cells of human, mouse, Chinese hamster and sea urchin. On immunoblots MPM-13 recognized a major protein band at 43×103Mr and a variable minor band at 56 ×103Mr in both mitotic and interphase HeLa cells. These antigens were detectable in a variety of mammalian cells as well as in the unicellular ciliate Tetrahymena. In cells arrested in mitosis by colcemid and stained with MPM-13 by indirect immunofluorescence, numerous fluorescent speckles were seen throughout the cytoplasm. Reversal of colcemid block in Chinese hamster ovary (CHO) cells by washing resulted in the reappearance of a fluorescent patch at the poles of the re-formed spindles. In HeLa cells arrested in mitosis by the microtubule stabilizing drug taxol, MPM-13 stained a large fluorescent patch encircled by dark metaphase chromosomes. This pattern indicated the failure of centrioles to move to the opposite poles in the presence of taxol. These data indicate that the MPM-13 antigens are associated with the colcemid-sensitive pericentriolar material from which microtubules originate but not with the centrioles themselves. It is also clear that these antigens are highly conserved during evolution.
Expression in Escherichia coli of fragments of glial fibrillary acidic protein: Characterization, assembly properties and paracrystal formation
Authors: Quinlan Roy A. , Moir Robert D. , Stewart Murray
Published: April 2021

We have expressed in Escherichia coli a 1258 bp cDNA fragment corresponding to 97% of mouse glial fibrillary acidic protein (GFAP), the principal intermediate filament protein of astrocytes. High levels of expression were obtained, as a fusion protein with 32 residues of the bacteriophage Â ell protein, using the pLcII expression vector system of K. Nagai and H.-C. Thogersen. Although removal of the cH protein fragment by proteolysis using factor X proved difficult, a protein corresponding to most of the cDNA fragment was obtained by cleaving at the endogenous thrombin site near the middle of the N-terminal non-helical domain of GFAP. A shorter 1047 bp fragment, in which the C-terminal non-helical domain of GFAP was deleted, was also produced using oligonucleotide-directed sitespecific mutagenesis of the original cDNA clone. After proteolysis with thrombin, this material gave a fragment that corresponded to the a-helical coiled-coil rod region of the GFAP molecule, together with a portion of the non-helical N-terminal domain. The fragments produced were characterized both biochemically and ultrastruc-turally, and appeared to retain the conformation of native GFAP. Crosslinking showed that all fragments formed molecules containing two chains (‘dimers’) that associated to form four-chain molecular dimers (‘tetramers’) analogous to those formed by intact intermediate filament proteins. Shadowed preparations showed the presence of rod-like particles that closely resembled those observed for other intermediate filament proteins and proteolytically prepared rod domains. Remarkably, the fusion protein produced from the entire 1258 bp cDNA fragment and the ell peptide was able to form filaments that closely resembled those produced by native GFAP. However, fragments in which either the cH peptide or the C-terminal nonhelical domain were removed, or in which both were removed, failed to form filaments under standard assembly conditions. Although preliminary in nature, these results suggest that both N- and C-terminal non-helical domains may have a role in intermediate filament formation. Moreover, the fragment corresponding approximately to the GFAP rod formed paracrystals similar to those observed with other coiled-coil proteins. The molecules in these paracrystals were arranged antiparallel with the two molecules in the unit cell, which may correspond to the Jour-chain molecular dimer (tetramer), overlapping by approximately two-thirds of their length.
Molecular cloning of the cDNA for ligatin
Authors: Jakoi Emma R. , Brown Alison L. , Ho Ye-Shih , Snyderman Ralph
Published: April 2021

We describe the first isolation and sequence of a partial cDNA clone encoding ligatin, a trafficking receptor for phosphoglycoproteins. The clone was isolated from a human U937 promonocyte λgtll cDNA library using rabbit antiserum to rat ileal ligatin. RNA blot hybridization revealed that the intact receptor transcript in human cells is 2-4 kilobases (kb). DNA sequencing together with expression of protein fusion products in Escherichia coli demonstrated that the cloned segment begins with a 1·2 kb open reading frame potentially encoding a 7·5×10 Mr section of the 10×103 Mr receptor followed by a 3′ tail of 948 bases. The 225 bases of coding sequence correspond to the carboxyl region of ligatin and contain a potential acceptor site for asparagine-linked glycosylation. Neither a poly(A) sequence nor polyadenylation signal was found at the 3′ end of the clone. In the 3′ untranslated region there is a paired consensus sequence that is analogous to a conserved 12 base-pair sequence present in clusters in several growth-controlled mRNAs, including those for c-fos and β-actin. The identity of this clone as ligatin was confirmed immunologically using antisera to an encoded fusion protein and three independent regions of its derived sequence. In addition, one of these antibodies produced a punctate immunofluorescence pattern within the cytosol of U937 cells in a similar fashion to anti-ligatin serum.
Changes in microvilli and golgi-associated membranes of lepidopteran cells induced by an insecticidally active bacterial δ-endotoxin
Authors: Lane Nancy J. , Harrison J. B. , Lee W. M.
Published: April 2021

The δendotoxin from Bacillus thuringiensis var. kurstaki was fed to late larvae of the tobacco hornworm, Manduca sexta, to determine its effect on the anterior midgut epithelium, which was examined ultrastructurally at intervals thereafter. The cells of the midgut are primarily of the columnar and goblet variety and the effects of the toxin were more pronounced on the former. Tissues examined after only 1·5 min of exposure to the toxin already revealed fine-structural alterations. These were most notably changes in the microvilli and membranes associated spatially with the Golgi complex: vacuoles associated with its maturing face became enlarged. This effect was intensified with more-extensive exposure to the toxin, resulting in an increase in both vacuoles and the number of lysosomal bodies, many containing myelin-like formations; some of these arose as autophagicvacuoles. There seemed to be no consistent increase in endocytotic activity at the apical border, however. The intramembranous particle population of the microvilli of the columnar cells showed some slight changes with toxin treatment; alterations in microvillar contours also occured. The intercellular septate and gap junctions on the lateral borders were sometimes disrupted and with time often became internalized. It seems, then, that the toxin initially modifies the microvillar membranes and subsequently the Golgi-associated saccules are affected, giving rise to vacuoles and lysosomes.
Computer-generated three-dimensional representation of dictyosomes reconstructed from electron micrographs of serial ultrathin sections
Authors: Kristen U. , Lockhausen J. , Menhardt W. , Dallas W. J.
Published: April 2021

Computer-generated three-dimensional reconstructions of higher plant dictyosomes from electron micrographs are presented from different viewing angles in the form of shaded perspective displays. The image processing has revealed complicated structures indicating a central cisternal stack surrounded by a cloud of vesicles and vesicular and tubular protrusions. The entire dictyosome is suggested as being an approximately spherical body that is marginally penetrated by plates and tubules of the endoplasmic reticulum.
Colony-stimulating factor-1 induces rapid behavioural responses in the mouse macrophage cell line, BAC1.2F5
Authors: Boocock C. A. , Jones G. E. , Stanley E. R. , Pollard J. W.
Published: April 2021

The cloned, SV40-immortalized mouse macrophage cell line, BAC1.2F5, resembles primary macrophages in its dependence on colony-stimulating factor-1 (CSF-1) for both viability and proliferation. Re-addition of CSF-1 stimulates rapid, transient behavioural changes in starved cells, which are rounded, with diffusely organized F-actin and few intracellular vesicles. Within 1 min, cells begin to spread, forming prominent, F-actin-rich ruffles. Small vesicles (0·5–1·0μm), formed throughout extending lamellar processes, move centripetally and, after 3–5 min, fuse to form larger vesicles (2·0–4·0μm), clustered around the nucleus. Immunofluorescence demonstrates that CSF-1, bound to cell-surface receptors, is internalized via these vesicles. Cell spreading and ruffling peak about 5 min after restimulation. Interference reflection microscopy indicates no corresponding change in the mode of cell-substratum adhesion: a single area of close adhesion underlies most of the cell and simply broadens during spreading. Analysis of cell aggregation kinetics shows no effect of CSF-1 on intercellular adhesiveness. Measurement of cell areas after starvation and restimulation demonstrates quantitatively the time-course and concentrationdependence of cell spreading. Mean area doubles within 5 min and, after a transient peak, decreases within 30 min to the value measured before starvation. This time-course corresponds to that of CSF-1 internalization and of the phosphorylation and subsequent degradation of CSF-1 receptors. The concentration-dependence of the spreading response resembles that of CSF-1-dependent survival and proliferation. The minimum detectable stimulation of spreading occurs at the concentration (22 pM) that supports survival without proliferation. Increasing stimulation of spreading occurs over the range of concentrations that elicit increasing proliferation.
Bimodal redistribution of surface transmembrane glycoproteins during Ca2+-dependent secretion (acrosome reaction) in boar spermatozoa
Authors: Aguas Artur P. , Da Silva Pedro Pinto
Published: April 2021

We used the acrosome reaction of boar sperm cells to study the dynamics of surface transmembrane glycoproteins (TMG) during a secretory process. The acrosome reaction is the Ca2+-dependent fusion of a large cytoplasmic vesicle (the acrosome) with the overlying segment of the plasma membrane (acrosomal cap) that leads to the release of the acrosomal enzymes. After triggering the acrosome reaction in vitro (2 mM-CaCU in the presence of 10μM-A23187), we used freeze-fracture electron microscopy to follow the topographical rearrangement of a population of acrosomal-cap large intramembrane particles that correspond to transmembrane proteins that bind wheat germ agglutinin. We found that these TMG move in the direction of either one of two opposite poles, proximal and distal, of the acrosomal cap. This bimodal movement of the TMG reorganizes the acrosomal cap into three extensive domains. The first two, on the apical rim and on the equator, are membrane domains to which the TMG are directed and where they accumulate. The third, a large in-between area of protein clearing, corresponds to the region from which TMG were preferentially located before displacement induced by the Ca2+ effect. The topography of these new membrane domains of the acrosomal cap becomes coincident with that of the structural domains of the subjacent acrosomal membrane. Mirroring of the acrosomal membrane by the plasma membrane is followed by fusion between the two membranes, formation of an exquisite labyrinth of hybrid-membrane tubules, followed by fission and release of the acrosomal contents through intertubular fenestrae.
A non-connexon protein (MIP) is involved in eye lens gap-junction formation
Authors: Gruijters W. T. M.
Published: April 2021

New immunolocalization data put the role of the lens MP26 (MIP) protein in a new perspective. During maturation of lens fibre cells, MIP is found to associate specifically with two structures, gap junctions and cell interlocking processes (known as ball and socket domains). It is significant that the zone in which these associations are most striking is discrete, coinciding with the zone of rapidly enlarging junctional plaques and newly forming ball and socket domains. Observation of domain-specific interactions of MIP with forming gap junctions and ball and socket domains suggests that MIP may be involved in the formation of close membrane appositions. Furthermore, previous ambiguities in the literature over the presence of MIP in gap junctions are clarified by the knowledge that, in situ, MIP associates strongly with gap junctions for only a brief period (with less than about 5 % of all lens gap junctions at any one time) during the assembly of junctional plaques.
Differential expression of membrane sialoglycoproteins in exudate and resident mouse peritoneal macrophages
Authors: Rabinowitz Stephen , Gordon Siamon
Published: April 2021

Wheat germ agglutinin (WGA) has been used to define biochemical differences between exudate and resident macrophages in the peritoneal cavity of mice. Western blotting with 125I-WGA identifies a restricted set of glycoproteins in elicited peritoneal macrophages (MΦ) (recruited with thioglycollate or periodate) and fully activated MΦ (recruited with live Bacille Calmette-Guérin, or heat-killed Corynebacterium parvum); the major species migrates with Mt 89-115 kDa in 10 % acrylamide gels. These glycoproteins are not detected in resident peritoneal MΦ, nor in thymocytes, neutrophils, lymphocytes and a variety of non-MΦ cell lines.

The binding of WGA is sensitive to neuraminidase, which exposes binding sites on these proteins for peanut agglutinin and reduces their electrophoretic mobility; these features are typical of O-linked sialo-oligosaccharides. In culture, exudate MΦ increase their WGA-binding content over 48 h, and continue to display a phenotype distinct from that of resident peritoneal MΦ.

The stable differential expression of these sialoglycoproteins, by elicited and activated versus resident peritoneal MΦ, suggests that biochemical modification during the synthesis and expression of membrane glycoproteins accompanies MΦ recruitment to an inflammatory focus.
Regulatory subunits of cyclic AMP-dependent protein kinase: presence in granules and secretion by exocrine and endocrine cells
Authors: Hand A. R. , Mednieks M. I.
Published: April 2021

Cyclic AMP-dependent protein kinase (cAPK) is the intracellular mediator of signal transduction events involving the adenylate cyclase-cyclic AMP system. A monoclonal antibody (MAb BB1) to the type II regulatory subunit (RII) of cAPK was used in a post-embedding immunogold-labeling procedure to determine the ultrastructural localization of RH in several different secretory cells of the rat. Label was present in nuclei, especially over the heterochromatin, and in the cytoplasm, particularly in areas containing rough endoplasmic reticulum. Immunolabeled RH was also present in secretory granules of the parotid gland, exocrine and endocrine pancreas, seminal vesicle, anterior and intermediate pituitary, and intestinal endocrine cells. Photoaffinity labeling of parotid saliva, pancreatic and seminal fluids with the cyclic AMP analogue, 32P-labeled-8-azido-cyclic AMP, revealed the presence of cyclic AMP-binding proteins with electrophoretic mobilities similar to those of authentic cAPK regulatory subunits. These results confirm our previous observations on the localization of cAPK regulatory subunits in the rat parotid using polyclonal antibodies, and extend them to a number of other exocrine and endocrine cells. The apparent widespread occurrence of cAPK subunits in secretory granules and secretory fluids suggests that cAPK may be involved in specific intragranular regulatory and/or phosphorylation events, or that it has an unidentified extracellular function.
Immunological detection of spectrin during differentiation and in mature ciliated cells from quail oviduct
Authors: Chailley Bernadette , Frappier Thierry , Regnouf Franqoise , Laine Marie-Christine
Published: April 2021

A protein that was immunologically related to the erythrocyte and brain α-240-subunit and to the brain β-235-subunit of spectrin was characterized by immunoblotting and was detected by immunofluorescence in the apical part of ciliated cells from quail oviduct. After immunogold-labeling electron immunocytochemistry, spectrin was detected mainly in a fibrillar meshwork located between the proximal parts of the basal bodies. It was also observed to be in contact with the basal foot of basal bodies, but was not found to be associated with the apical plasma membrane. Cilia and microvilli were unlabeled. In contrast, spectrin was detected in close contact with the lateral plasma membrane of mature ciliated cells as well as in stem epithelial cells in unstimulated oviduct. During ciliogenesis induced by estrogen, spectrin gradually appeared at the apex of the cells as the apical cytoskeleton differentiated.
Nerve growth in the absence of growth cone filopodia and the effects of a small applied electric field
Authors: Mccaig Colin D.
Published: April 2021

Nerve orientation may involve a biasing of the distribution of tension at the growth cone. Chemical and electrical guidance cues cause more filopodia to appear on one side of the growth cone and this may determine turning behaviour. In a small applied electric field, filopodia predominate on the cathodal side of the growth cone and nerves turn towards the cathode. Removing all filopodia by treatment with cytochalasin D did not prevent nerves from continued slow growth and nerves still oriented towards the cathode. It is concluded that nerves can perform some types of orienting behaviour in the complete absence of filopodia.
Eurythermic and stenothermic growth of cultured fish cells and their thermosensitivity
Authors: Mitani Hiroshi , Naruse Kiyoshi , Shima Akihiro
Published: April 2021

RBCF-1 cells derived from the caudal fin of the goldfish (Carassius auratus) have been established at 37°C, and could grow at a wide range of temperatures (20-37°C). To examine the mechanism(s) of this eurythermic property, we isolated clones from RBCF-1 cells with different optimal growth temperatures. R37 clone grew continuously at 37°C but could not grow at 27°C. R27 clone grew continuously at 27°C but could not grow at 37°C. Thus R37 and R27 cells may be referred to as stenothermic. The R3727 clone, which was derived from R37 cells selected at 27°C, and the cell hybrids of R37 and R27 cells, could proliferate for a long time and form colonies at 27–37°C, like progenitor RBCF-1 cells, so they may be referred to as eurythermic. The shape of colonies of R3727 cells changed with incubation temperature; they resembled those of R37 cells at a higher temperature (37°C), while at a lower temperature, (27°C), they were like those of R27 cells.

The lethal effects of heat treatment were compared among RBCF-1 clones. R27 cells were most sensitive, and R37 cells were most resistant. The heat treatment could induce the four major heatshock proteins (hsp30, hsp42, hsp70 and hsp90). Marked induction of hsp70 and hsp30 was observed at 33°C for R27, at 37°C for R3727 and at 40°C for R37. With R3727 cells, induction of hsps and thermosensitivity were highly dependent on the incubation temperature before heat treatment.
Confocal scanning optical microscopy and its applications for biological specimens
Authors: Shotton David M.
Published: April 2021

Confocal scanning optical microscopy (CSOM) is a new optical microscopic technique, which offers significant advantages over conventional microscopy. In laser scanning optical microscopy (SOM), the specimen is scanned by a diffractionlimited spot of laser light, and light transmitted or reflected by the in-focus illuminated volume element (voxel) of the specimen, or the fluorescence emission excited within it by the incident light, is focused onto a photodetector. As the illuminating spot is scanned over the specimen, the electrical output from this detector is displayed at the appropriate spatial position on a TV monitor, thus building up a two-dimensional image.

In the confocal mode, an aperture, usually slightly smaller in diameter than the Airy disc image, is positioned in the image plane in front of the detector, at a position confocal with the in-focus voxel. Light emanating from this in-focus voxel thus passes through the aperture to the detector, while that from any region above or below the focal plane is defocused at the aperture plane and is thus largely prevented from reaching the detector, contributing essentially nothing to the confocal image. It is this ability to reduce out-of-focus blur, and thus permit accurate non-invasive optical sectioning, that makes confocal scanning microscopy so well suited for the imaging and three-dimensional tomography of stained biological specimens.

In this review, I explain the principles of scanning optical microscopy and blur-free confocal imaging, discuss the various imaging modes of confocal microscopy, and illustrate some of its early applications.
The localization of the divergent β2-tubulin isotype in the microtubular arrays of Physarum polycephalum
Authors: Diggins-Gilicinski Maureen , Solnica-Krezel Lilianna , Burland Timothy G. , Paul Eileen C. A. , Dove William F.
Published: April 2021

The β2-tubulin isotype of Physarum polycephalum is only 83 % identical in amino acid sequence with the constitutively expressed β1B-tubulin and the myxamoeba-specific βlα-tubulin isotypes. A poly-clonal antibody specific for β2-tubulin was used to monitor the subcellular distribution of the β2-tubu-lin antigen in the mitotic spindle of the mature plasmodium – the sole microtubular array in that stage of Physarum. By immunofluorescence, the β2-tubulin antigen was detected throughout this an-astral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No β2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the β2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial β2-tubulin isotype in a mitotic spindle does not determine a closed, anastral mitosis.
Microtubules rich in modified α-tubulin characterize the tail processes of motile fibroblasts
Authors: Prescott A. R. , Vestberg M. , Warn R. M.
Published: April 2021

The organisation of microtubules rich in post-transiationaily modified α-tubulin has been investigated in a fibroblast cell line (NIH–3T3–T15) that can be reversibly transformed. An immunofluorescence microscopy study of the static non-trans-formed cells has revealed a central distribution of wavy microtubules showing post-translational modifications. When transformed there is a marked increase in cell motility and the appearance of long thin cytoplasmic ‘tails’. These tails have been found to contain conspicuous bundles of post-trans-lationally modified microtubules that run down the length of the processes and terminate close to the plasmalemma. Both detyrosinated and acetylated α-tubulin are present as major species in these modified microtubules. Such a pattern of modified microtubules is only occasionally seen in the untransformed NIH–3T3–T15 cells. We have also found them to be present in other transformed fibroblast lines. The presence of bundles of micro-tubules rich in modified α-tubulin in the cell tails is correlated with a marked reduction in the numbers of F-actin stress fibres. The possible role of these modified stable microtubules in cell motility is discussed.
The fate of the centrosome–microtubule network in monocyte-derived giant cells
Authors: Moudjou Mohammed , Lanotte Michel , Bornens Michel
Published: April 2021

Avian monocyte-derived giant cells in vitro, which are in many respects similar to osteoclasts, display a complex microtubule array that plays a prominent role in cell spreading. It is organized by a polygonal row of regularly spaced centrosomes surrounding an irregular cluster of nuclei. The immediate progenitor cells are binucleate cells with a single microtubule- organising center (MTOC), the result of the congregation of the two individual centrosomes. The one-to-one correspondence between numbers of centrosomes and nuclei in giant cells suggests that the centrosome of each precursor cell has been conserved through the fusion process. This is in marked contrast to the absence of centrosomes in myotubes, another example of a differentiated cell derived from the fusion of progenitor cells.
Stringency at four regions of the plant cell cycle where proteins regulating its progression are synthesized*
Authors: torre C. De la , Gonzalez-fernandez A. , Gimenez-martin G.
Published: April 2021

Different responses were detected when inhibiting protein synthesis (by 1μg ml-1 anisomycin) for similar times (3 h) in each of the four regions of the plant cell cycle where synthesis of specific proteins seems to control further cycle progression. Synchronous cells, labelled as binucleate by 1-h, 5 mM-caffeine treatment, were used for this study in Allium cepa L. root meristems proliferating under steady-state kinetics.

In the experimental conditions used, G1 lasts 8 h in the synchronous control populations. G1 houses two such regions. The first region is located in early G1. Inhibition of protein synthesis in this region produced 8·2 h of delay in reaching the S period. Moreover, the treatment halved the prolUerative fraction of the population, behaving as a restriction area where cells should be rendered competent to go on cycling by newly synthesized proteins. The second region, located in late G1 (before the 6th hour of the cycle), produced, when inhibiting protein synthesis in the population, a delay of 2·4h (shorter than the treatment) in reaching the S period and no decrease of its proliferative fraction.

A similar inhibition of protein synthesis in the third region mapped in early G2 (before the 20th hour of the cell cycle) produced 4·4h of delay in reaching mitosis and a 20 % decrease in the proliferative fraction. Finally, there is a fourth region in early prophase (before the 25th hour of the cell cycle) where all the cells seemed to abandon the cycle during inhibition of protein synthesis.
Three-dimensional analysis of the organization of human chromosome domains in human and human-hamster hybrid interphase nuclei
Authors: Dekken Herman Van , Pinkel Daniel , Mullikin Jim , Trask Barbara , Engh Ger Van Den , Gray Joe
Published: April 2021

This report describes the intranuclear organization of chromosomes in human–hamster hybrid nuclei and in human cell nuclei. The target chromosomes were stained using in situ hybridization with biotin-ylated, chromosome-specific DNA probes. Bound probe was detected with fluorescein–avidin. Hybridizations were performed to fixed nuclei in aqueous suspension in order to preserve their threedimensional morphology. Total nuclear DNA was stained with DAPI. Three-dimensional information about the organization of DNA and probe within the nucleus was obtained by optical sectioning.

The human chromosomes in human–hamster hybrid nuclei were found to be confined to ‘domains’ that were maintained during the cell cycle. Different spatial localization patterns of the human chromosomes were seen in interphase nuclei of two different hybrid cell lines. The positions of chromosome-specific repetitive sequences in human fibroblast interphase nuclei were also studied using probes for the telomeric region of chromo-some Ip (Ip36), the centromeric region of chromosome 9 (9ql2) and the long arm of the Y chromosome (Yql2). These studies showed that the two lρ telomeric loci are located near the nuclear surface. The chromosome 9 centromeric loci are similarly located. Simultaneous hybridization of the chromosome 1 telomeric probe (target size ∼200kb; b, base) and the Y-specific probe (target size >2Mb), demonstrate that the binding sites of the two probes can be distinguished in the same nucleus on the basis of domain size.
A somatic cell-derived system for studying both early and late mitotic events in vitro
Authors: Nakagawa Junichi , Kitten Gregory T. , Nigg Erich A.
Published: April 2021

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone Hl ; moreover, they specifically contain four soluble phosphoproteins of Mr 38 000, 75 000, 95 000 and 165 000. Following disassembly of exogeneous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.
Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos
Authors: Harel Amnon , Zlotkin Efrat , Nainudel-Epszteyn Sandra , Feinstein Naomi , Fisher Paul A. , Gruenbaum Yosef
Published: April 2021

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, gpl88, 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 embryo-genesis in Drosophila. This ‘spindle envelope’, as it has been named by others, contains both lamin and otefin but probably not pore complex proteins.
A new monoclonal antibody to a cell-surface antigen that distinguishes luminal epithelial and myoepithelial cells in the rat mammary gland
Authors: Mahendran R. S. , O’Hare M. J. , Ormerod M. G. , Edwards P. A. W. , McIlhinney R. A. J. , Gusterson B. A.
Published: April 2021

A monoclonal antibody (25.5) has been produced that recognises luminal epithelial cells of the rat mammary gland. This antibody together with monoclonal anti-CALLA antibodies, which react with mammary myoepithelial cells, has been used in biochemical, immunocytochemical and flow cytometric studies. Antibody 25.5 bound to proteins of molecular weight 70K and 25K (K=103Mr) in both the rat milk fat globule membrane and in single cell suspensions prepared from the virgin adult rat mammary gland. Anti-CALLA antibody (J5), recognised a 93-100K protein in the gland extracts, which co-electrophoresed with the CALLA/CD-10 antigen from NALM-6 acute lymphoblastic leukaemia cell line.

Antibody 25.5 bound to the luminal surface of rat mammary epithelial cells at all stages of development from neonatal through to pregnancy, lactation and involution. CALLA immunoreactive staining has previously been shown on basally located pre-sumptive myoepithelial cells at all stages of development. Flow cytometric analyses demonstrated that 25.5 and anti-CALLA antibodies stained independent cell populations in suspensions of single cells prepared from purified epithelial elements from the mammary gland of adult virgin rat.
The rna content of the nucleolus and nucleolus-like inclusions in the anther of Lilium estimated by an improved rnase-gold labelling method
Authors: Sato S. , Willson C. , Dickinson H. G.
Published: April 2021

Using RNase-gold labelling a comparative estimation has been made of the RNA content of the nucleofi and nucleofus-like inclusions in anther cells of Lilium henryi Thunb. Pretreatment with glow electric discharge prior to application of the RNase-gold labelling remarkably lowered the level of nonspecific adsorption and allowed specific labelling of RNA-containing structures. In the tap-etal cells, the nucleofi, the perichromatin material and the dense bodies labelled heavily, but both the interchromatin region and the condensed chromatin did not bind the label. The nucleofus-like inclusions, which were found in the nucleoplasm of meiotic cells at the tetrad stage, and were termed‘nuclear nucleofus-like bodies (NLBs)’ in the present study, also showed a high response compared with both the loosened and the condensed chromatin; this labelling was some 60% of that observed over the nucleofi of somatic cells. Another type of nuclear NLB was differentiated from the predominant type of NLB by a conspicuous electron opacity, reduced size, and strong labelling with the RNase-gold complex, suggesting that some nuclear NLBs may be highly condensed. The labelling over the cytoplasmic nucleofoids (nucleofus-like inclusions found in the cytoplasm) was only 50 % of that over the nuclear NLBs, although both are similar in ultrastructural texture.
Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals
Authors: Buchanan Janet A. , Yeger Herman , Tabcharani Joseph A. , Jensen Timothy J. , Auerbach Wojtek , Hanrahan John W. , Riodan John R. , Buchwald Manuel
Published: April 2021

We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori−. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMPdependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.
Cell studies of total internal reflection fluorescence: Effect of lipid membranes
Authors: Heavens O. S.
Published: April 2021

The intensity of the fluorescence generated when cell-substratum contacts are studied by total internal reflection fluorescence is calculated. In previous work, some approximations were made in respect of the effect of the lipid membranes. These approximations are removed in the present treatment.
Interphase microtubule dynamics are cell type-specific
Authors: Wadsworth P. , Mcgrail M.
Published: April 2021

The rate and pattern of microtubule polymer loss in interphase cells have been examined using nocod-azole to block microtubule assembly. Cells were incubated with high concentrations of nocodazole for various times and the pattern of microtubule disassembly was determined using tubulin immunofluorescence. Polymer loss was quantitated by measuring the decrease in percentage of cell area occupied by microtubules. The results demonstrate that microtubules in diverse cells disassemble individually and asynchronously. In addition, these quantitative measurements reveal that epithelial and fibroblast cells display strikingly different kinetics of polymer loss. In fibroblasts, polymer loss is rapid, with a half-time of 4 min at 37°C. In epithelial cells, loss of 60% of the microtubules occurs with a half-time of 18 min; the remaining 40% of the microtubules disassemble much more slowly (average half-time of 72 min). To demonstrate that these differences were not due to species differences among various cells assayed in these experiments, epithelial and fibroblast cells derived from primary cultures of newt lung have been examined. Again, fibroblast and epithelial cell microtubule dynamics could be readily distinguished. To determine if modifications to epithelial cell microtubules contribute to their stability, microtubules were completely disassembled and allowed to regrow. The rate of polymer loss for recently regrown microtubules was more rapid than microtubules in control cells, indicating that stability increases with time after assembly.