bluuebunny/crossref_metadata_2025 huggingface dataset:
The ultrastructure and ontogeny of pollen in Helleborus foetidus l. iv. pollen grain maturation
Authors: Echlin P.
Published: April 2021

The final stages of Helleborus pollen-grain ontogeny, which culminate in maturation and germination of the grain, have been investigated at the ultrastructural level. Following the deposition of primary and secondary exine, and during the early stages of intine formation, the microspore passes through a vacuolate phase, in which the cytoplasm appears devoid of most organelles other than the prominent nucleus. The formation of the vacuole results in the displacement of the nucleus to one side of the pollen grain. The vacuole quickly disappears and a number of organelles reappear in the cytoplasm, in particular the dictyosomes and strands of endoplasmic reticulum, with associated grey bodies. Following mitotic division of the pollen grain, the first signs of the generative cell wall appear as a pair of tightly appressed unit membranes in the narrow strand of cytoplasm separating the two newly formed generative and vegetative nuclei. As development proceeds, the space between the two membranes gradually fills with an electron-transparent material similar to the substance found in the numerous dictyosome-derived vesicles which, together with the endoplasmic reticulum, are both closely associated with the developing cell wall. The generative cell wall fuses with the cellulosic intine, which has gradually increased in amount during these stages, and the cell division is complete. The smaller generative cell contains a prominent nucleus and a small amount of cytoplasm devoid of plastids and most other organelles. The larger vegetative cell also contains a prominent nucleus and a large amount of cytoplasm containing amyloplasts, mitochondria, dictyosomes and endoplasmic reticulum, and abundant ribosomes, many of which are in a polysome configuration. The final stages in development are characterized by a progressive decrease in the amount of starch in the vegetative cell and an increase in the size of grey bodies, many of which are invested in multilayered shrouds of endoplasmic reticulum. The generative cell wall disappears and a multivesicular/granular body gradually appears at the periphery of the pollen grain. The granular-vesicular material, which is formed from the dictyosomes and/or the degenerating plastids, is thought to represent metabolic reserves necessary for pollen-tube formation. One or more pollen tubes emerge from the apertural sectors of the pollen grain, and maturation of the grain is complete.
Isolation and characterization of chromatin and dna from the sperm of the spider crab, Libinia emarg1nata
Authors: Vaughn Jack C. , Hinsch Gertrude W.
Published: April 2021

An isolation technique is described which yields highly purified sperm from various regions of the reproductive tract of the crab Libinia emarginata. Sperm chromatin was isolated and analysed with respect to ultrastructure, ultraviolet absorption spectrum, thermal melting profile, behaviour in CsCl gradients, and chemical composition. Sperm-isolated chromatin not stabilized with uranyl acetate prior to ethanol dehydration, like prokaryotic chromatin, has aggregated fibres with a diameter in the range 30–95 nm or more. With uranyl acetate stabilization, only very fine fibres are seen. Pronase treatment of the thick fibres leads to formation of very fine fibres, showing that protein is associated with this isolated chromatin. Polyacrylamide electrophoretic analysis of the chromatin protein shows that it is not basic. Standard basic protein staining of sperm taken from testes, vas deferens, and seminal receptacles after various periods of storage there gives a negative nuclear reaction in all cases. This suggests the absence of sperm nuclear histones and protamines. Sperm chromatin protein is salt-dissociable and has a ratio of basic to acidic amino acids of 0·43–0·48. The-protein/DNA ratio is 2·3–3·2, and the ratio E280/E260, is 0·50–0·67. Thermal denaturation curves are identical for sperm chromatin and purified sperm DNA, supporting the other data showing lack of nuclear basic proteins. An (A + T)-rich satellite DNA component comprises about 7–9 % of the total sperm genome. It is suggested that the genes which specify gamete-type nuclear basic proteins are either permanently repressed or deleted in decapod Crustacea.
Isolation of homologous nuclear dnas from sea-urchin embryos
Authors: Baker R. F.
Published: April 2021

Studies are presented on the ability of low-molecular-weight nuclear DNA fractions to hybridize to higher-molecular-weight nuclear DNAs taken from different stages of early developing sea-urchin embryos, Strongylocentrotus purpuratus. Using preparative DNA-DNA hybridization, a fraction of 60-s mid-blastula DNA was isolated for its ability to anneal to 10-s morula DNA. Approximately 80 regions on each 60-s molecule were found to be homologous to 10-s DNA. High-molecular-weight (> 240-s) nuclear DNA from mesenchyme blastula stage and later stages (compared with DNA from pre-blastula nuclei) shows an increase in the number of regions homologous to fractions of 10-and 60-s DNAs.
A scanning electron-microscope study of the surface features of mammalian cells in vitro
Authors: Hodges Gisele M. , Muir Marjorie D.
Published: April 2021

The cell surface aspects and the density, distribution and morphology of surface cytoplasmic projections (microprocesses) of HeLa, BHK and baby mouse kidney cells, maintained under different culture conditions, have been compared using scanning electron microscopy. No significant differences were noted in the surface aspects of these cell strains. Comparison of the 3 cell strains, under identical culture conditions, showed variations from one strain to another in the density and morphology of the cell surface microprocesses which may reflect basic physiological differences between the strains. For a given cell strain, variations were observed in response to changes in culture conditions indicative that the presence of surface microprocesses is related to the physiological state of the cell.
Absence of metabolic co-operation between certain types of mammalian cells in culture
Authors: Widmer-Favre Chantal
Published: April 2021

A mutant line of BHK21 cells lacking inosinic pyrophosphorylase activity (IPP− cells) does not incorporate [3H]hypoxanthine, as opposed to wild type BHK cells. When IPP+ and IPP− cells are grown together in the presence of [3H]hypoxanthine, IPP− cells in contact with IPP+ cells incorporate the marker, as seen by autoradiography. This phenomenon is known as ‘metabolic co-operation’.

In the present work no co-operation was detected between wild type BHK cells and BHK cells lacking thymidine kinase activity (TK− cells – they do not incorporate [3H]thymidine); between wild type L cells and L-TK− cells, or between L cells and L-IPP− cells. And there is no interaction in mixed cultures of mast cells and mast-cells-IPP−.
Multiple symbiosis in a leafhopper, Helochara communis fitch (cicadellidae: Homoptera): Envelopes, nucleoids and inclusions of the symbiotes
Authors: Chang K. P. , Musgrave A. J.
Published: April 2021

Mycetomes in Helochara communis are paired syncytia each enclosed by an epithelium. Adjacent are loose aggregates of mycetocytes. Mycetomes harboured 3 kinds of inclusions: companion, a-and t-symbiotes according to Buchner’s classification of homopteran symbiotes. In the mycetocytes, a fourth kind of inclusion was found and named g-symbiotes. All inclusions were Feulgen and Gram negative. Nucleoids were revealed in companion symbiotes by osmium-HCl-Giemsa; and, in some g-and t-, but not in a-, symbiotes by RNase-papain Giemsa. DNase digestion caused loss of basophilia in g-and t-, but not in a-, symbiotes. By electron microscopy, using Kellenberger’s fixative, companion, g-and t-symbiotes had lucid areas with reticular strands 4 nm thick; a-symbiotes were in general, homogeneously densely granular. Companion symbiotes had Gram-negative profiles with additional surface material; a-and t-symbiotes had 3, g-symbiotes 2, peripheral, trilaminar membranes. Parallel striations and regularly arranged granules occurred in companion symbiotes; crystalline inclusions in g-and t-symbiotes; and fibrillar elements and dense bodies in a-symbiotes.

Companion symbiotes may be Gram-negative bacteria or rickettsiae; g-and t-symbiotes, L-phase bacteria; but a-symbiotes are anomalous, apparently lacking DNA, and probably being derivatives of t-symbiotes, thus requiring that Muller’s hypothesis of homopteran evolutionary pathways be rephrased.
A fine-structural study of conidium initiation in Stemphylium botryosum wallroth
Authors: Carroll Fanny E.
Published: April 2021

Conidia and conidiophores from synchronously sporulating cultures of Stemphyliiim botryosum were fixed and embedded at successive intervals for examination under the electron microscope. The spore initial arises as a small bud at the tip of the conidiophore, and no discontinuity between the conidiophore wall and the spore can be detected. The single-celled bud and the tip cell of the conidiophore have very similar cytoplasmic features.

Besides the usual cellular organelles, irregularly shaped electron-transparent areas in which membrane systems seem to perform some internal secretory activity between the leaflets them selves are present. Globules of the slightly electron-dense material originating in these specialized areas can also be found next to the plasma membrane where they are released and presumably participate in wall formation. Such a secretory system has not been described before and may be of particular importance in higher fungi which are known to lack a true Golgi apparatus.
Freeze-fracture replication of rough endoplasmic reticulum of mouse liver cells
Authors: Breathnach A. S. , Stolinski C. , Gross M.
Published: April 2021

Fresh, chemically unfixed, glycerinated specimens of mouse liver were examined by the technique of freeze-fracture replication without sublimation (i.e. they were not ‘etched’). Where extensive areas of fractured lamellar membranes of the rough endoplasmic reticulum are revealed en face, 2 types of fracture face are distinguishable. One of these fracture faces (A) is directed towards the cytoplasm, and the other (B) towards the cisternal cavity. A characteristic mosaic, or patchwork pattern of flat areas circumscribed by particles, is evident on both faces, and more clearly so on face B, due to a greater number of more prominent particles. Similar mosaic patterns are revealed on convex faces erf the nuclear membrane, and on concave fracture faces of mitochondrial membranes, but are not evident on fracture faces of the plasma membrane.

Uncertainty in establishing the exact plane of fracture of membranes in this material, since glycerol is virtually non-sublimable, makes it difficult to assess the significance of these mosaic patterns. The fact that ribosomes are not identifiable on either face of fractured endoplasmic reticulum membranes, gives no certain indication of the plane of fracture.
An actin-like component in sperm tails of a crane fly (Nephrotoma suturalis loew)
Authors: Forer A. , Behnke O.
Published: April 2021

In negatively stained preparations made from glycerinated crane fly spermatids and sperm, actin-like filaments are seen which bind heavy meromyosin (HMM) to form arrowhead complexes, the reaction with HMM being blocked by ATP and pyrophosphate. In preparations from young spermatids the actin-like filaments are found singly, or in small groups, while in those from mature sperm the actin-like filaments are organized into a structure which we call ‘rods’. Both rods and single filaments come from lysed sperm tails. The actin-like filaments in rods bind HMM only when frayed out on a grid.

In sections of normal or glycerinated spermatids or sperm, no actin-like filaments are seen, either because they are not preserved through the fixation and embedding procedures, or because they are present in a form which we do not recognize. In sections of glycerinated sperm-atids incubated with HMM, decorated filaments are seen in non-nuclear regions of spermatids (tails), oriented parallel to the axoneme. These probably correspond to the single filaments identified in the negatively stained preparations and not to the rods, because the actin-like filaments in rods bind HMM only after fraying out on a grid.

HMM causes polymerization of filaments: actin-like filaments are not seen in negatively stained preparations of glycerinated cells subsequently incubated in salts solution, yet are seen in such cells after a further incubation in HMM. Thus some of the decorated filaments seen in sections may have been polymerized by the HMM, raising questions about the procedure.

After rupture of living cells no single actin-like filaments are seen in negatively stained preparations, raising the question of whether glycerol as well as HMM can cause polymerization of filaments. Rods are seen in such preparations, and thus rods seem to exist as such in living cells.

Single actin-like filaments and rods are seen even when cytoplasmic microtubules are not seen (after incubation in colchicine or vinblastine). Thus the filaments do not seem to come directly from cytoplasmic microtubules.

The possible presence of actin-like filaments in cilia and in other flagella, the location of actin-like filaments in sperm tails, and the possible role of the actin-like filaments are discussed.
The basis of cell-to-cell transformation in Paramecium bursaria: II. investigation into the molecular nature of the transforming agent
Authors: Cullis C. A.
Published: April 2021

A mating type transformation occurring in syngen 4 of Paramecium bursaria has been further investigated. The transformation event was not reversed or prevented by acridine or ionizing radiations. The transformation has not been accomplished with cell-free extracts including the microinjection of cytoplasmic extracts.

The nucleic acids of normal and transformed cells have been compared. No differences were found. Both types had 2 major ribosomal RNAs of molecular weights 1·29 × 106 and 0·70 × 106 Daltons. In both types a particular RNA species, with molecular weight 10 × 105 Daltons, was found only in sexually competent cells. The DNA had a buoyant density in caesium chloride of 1·689 8 cm−3 and a Tm in 0·1 × SSC (1 × SSC = 0·15 M sodium chloride, 0·015 M trisodium citrate) of 65 2 °C. The renaturation kinetics showed 2 types of DNA – a fast-renaturing com ponent, comprising 13% of the total DNA, with a complexity of 1·84× 109 Daltons, and a slow-renaturing component with a complexity of r6xio u Daltons, in both normal and transformed cells. Possible models for the action of the transforming agent are discussed.
The Responses in Culture of Human Tumour Astrocytes and Neuroblasts to N6,O2′-Dibutyryl Adenosine 3′,5′-Monophosphoric Acid
Authors: Macintyre E. H. , Wintersgill C. J. , Perkins J. P. , Vatter A. E.
Published: April 2021

N6,O2′-dibutyryl cyclic AMP (DBcAMP) (3× 10-4 M) causes reproducible changes in the morphology, growth rate and terminal cell density of 3 different types of human tumour cells grown in culture.

Human tumour astrocyte lines 1181N1 and EH-118MG show generalized ablation of membrane systems when grown in the presence of this cyclic AMP analogue. This is not consistent with differentiation; it may be a toxic effect but possibly represents the response of these cell types to DBcAMP. These cells also grow more slowly and to lower terminal cell densities as a function of the concentration of DBcAMP. Although the growth kinetics demonstrate a return of these malignant cells to a state of density-dependent growth, micrographic analyses of intercellular relationships reveal that the cells still grow in overlapping patterns. Thus as judged by the commonly accepted criteria, DBcAMP does not induce contact inhibition of growth. All observed changes induced by the presence of DBcAMP are rapidly and completely reversed upon its removal from the growth medium. Human neuroblasts (NBI) grown in the presence of DBcAMP show irreversible morphological changes and the rate and extent of cell division is reduced. There is marked hyperplasia of intracytoplasmic microfilaments, which is considered consistent with differentiation.
Studies on Nucleolar RNA Synthesis in Drosophila Melanogaster: I. The Relationship between Number of Nucleolar Organizers and Rate of Synthesis
Authors: Krider H. M. , Plaut W.
Published: April 2021

Nucleolar RNA synthesis in salivary glands from XX, XY, and XO larvae having different numbers of nucleolus organizers was examined autoradiographically following incubation of (he tissues in [3H]uridine. In addition, the presence or absence of secondary constrictions was monitored in neuroblast preparations using acridine orange staining. It was observed that: (1) The rate of nucleolar [3H]uridine incorporation is independent of the number of nucleolus organizers in the cell; (2) nucleolar incorporation in XO tissues is elevated relative to that observed in XX or XY material; and (3) where the number of organizers is in excess of the wild type (2), secondary constrictions form at only 2 of the nucleolus organizer sites.

From these and related observations we suggest that there are 2 forms of transcriptional control for nucleolar RNA synthesis. One acts as an on-off control, influencing the formation of secondary constrictions at the nucleolus organizers. The other form modulates rates of transcription at organizers where constrictions have been formed.
The Nature of Immature Avian Erythrocytes in Severe Anaemia Induced by Phenylhydrazine
Authors: Williams A. F.
Published: April 2021

Hens were rendered severely anaemic by the use of phenylhydrazine and the characteristics of the polychromatic erythrocytes appearing in the blood examined. Even the least-dense polychromatic erythrocytes obtained after density fractionation of anaemic blood on BSA density gradients had a volume mode only 1-5 times that of mature erythrocytes. Also the immature cells always contained less haemoglobin than mature erythrocytes.

It was concluded that in severe anaemia avian erythropoiesis still consists of only one cell line, and that cells do not miss divisions as is believed to be the case in mammalian erythropoiesis.
Deoxythymidine Metabolism in Avian Erythroid Cells
Authors: Williams A. F.
Published: April 2021

The ability of dividing and non-dividing cells of the avian erythropoietic series to phosphorylate [3H]deoxythymidine and [3H]uridine has been studied. Enzyme levels of deoxythymidine kinase were also assayed. Both dividing and immature non-dividing erythroid cells phosphorylated uridine to uridine triphosphate (UTP). However, only cells able to synthesize DNA could phosphorylate deoxythymidine to the triphosphate; also these cells were the only ones to show detectable deoxythymidine kinase activities.
DNA Polymerase in Avian Erythroid Cells
Authors: Williams A. F.
Published: April 2021

The level and properties of DNA polymerase activity assayable in extracts of avian erythroid cells was studied. The enzyme was detectable in the dividing cells (erythroblasts) of the erythropoietic series and also the immature non-dividing erythrocytes. It could not be assayed in mature erythrocytes. Investigations showed that activity began to decline at the time of the last cell division of the erythroid series. Properties of the enzyme did change in different cell types; however, the changes did not correlate with cessation of DNA synthesis. Some preliminary results on DNA synthesis by isolated nuclei are also reported and these showed that only nuclei from erythroblasts could synthesize DNA in vitro in the absence of primer.
The Structure of Trichocysts in Paramecium Caudatum
Authors: Bannister L. H.
Published: April 2021

The structure of undischarged and discharged trichocysts has been examined in Paramecium caudatum, and their light-microscopic appearance compared with their fine-structural organization. In living specimens undischarged trichocysts appear to be of a single type with a unimodal variation in length about a mean of 3·7 μm. When fixed for electron microscopy or compressed beneath a coverslip many of the trichocysts expand within the cell, giving rise to a variety of different forms of lower phase density.

Ultrastructurally the undischarged trichocyst consists of at least 10 different components: these include a mesh-like sheath surrounding the body of the organelle; an inner and an outer sheath enclosing the tip, the inner sheath being made up of 4 spiralling envelopes with a square net substructure, and the outer sheath being formed of a dense amorphous matrix containing longitudinal microtubules and scattered fine filaments; a boundary surface to the outer sheath; a membranous trichocyst sac the apical region of which is surrounded by a cylinder of microtubules joined to each other with dense material; and lastly, the crystalline matrix of the trichocyst body and tip. This crystalline appearance is apparently related to the presence of a loosely interwoven complex of fine filaments which form a highly regular pattern of unit structures repeating at 16-nm intervals.

In extended trichocysts the 60-nm banding pattern of the body is also composed of fine filaments arranged in a different, elongated manner in 2 distinct and alternating patterns which are taken to be 2 views of the same structure. Measurements indicate that when trichocysts extend they elongate by a factor of from 6 to 8. It is proposed that the crystalline pattern of the unextended trichocyst body transforms into the extended form by a simple rearrangement of the constituent filaments accompanied by their elongation. Possible models of the undischarged and discharged states of organization are suggested.
Prenucleolar bodies contain coilin and are assembled in Xenopus egg extract depleted of specific nucleolar proteins and U3 RNA
Authors: Bell Peter , Scheer Ulrich
Published: April 2021

Nuclei assembled in Xenopus egg extract contain numerous spherical aggregations or nuclear bodies. Previously we have shown that they closely resemble prenucleolar bodies (PNBs), both at the compositional and ultrastructural level. Subsequently, coilin was also identified and for this reason they were called coiled bodies. Here we present morphological and immunocytochemical evidence that the in vitro nuclear bodies resemble authentic PNBs and are different from coiled bodies. In particular we show that coilin, previously considered as the defining protein constituent of coiled bodies, is also present in PNBs of cultured cells. In contrast, the PNB-associated nucleolar proteins nucleolin and B23/NO38 are not detectable in coiled bodies and may thus serve as suitable markers for PNBs. Our results suggest that PNBs are primary assembly structures which contribute to the formation of both nucleoli and coiled bodies and thus offer an explanation for the frequently observed structural association of coiled bodies with nucleoli. To gain some insight into the assembly process of PNBs in vitro, specific nucleolar proteins were removed from Xenopus egg extract. Quite surprisingly, the immunodepleted extracts still promoted the assembly of nuclear bodies which lacked either fibrillarin, nucleolin, xNopp180 or B23/NO38. Only after fibrillarin-depletion fewer PNBs were seen as compared to controls. Digestion of the extract with RNase followed by northern blot analysis revealed that U3 small nucleolar RNA is not required for the formation and structural maintenance of PNBs in vitro.
Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity
Authors: Helvoort Ardy van , Giudici Maria Luisa , Thielemans Marion , Meer Gerrit van
Published: April 2021

Sphingomyelin is a major lipid of the mammalian cell surface. The view that sphingomyelin, after synthesis in the Golgi lumen, reaches the outer leaflet of the plasma membrane on the inside of carrier vesicles has been challenged by inconsistencies in the results of transport studies. To investigate whether an alternative pathway to the cell surface exists for sphingomyelin, brefeldin A and mitotic cells were used to block vesicular traffic between the Golgi complex and the plasma membrane. Exogenous sphingomyelinase was applied in the cold to assay for the presence of sphingomyelin on the surface of CHO cells. Newly synthesized radiolabeled sphingomyelin was found to equilibrate with cell surface sphingomyelin within 1.5 hours at 37°C. Brefeldin A and mitosis inhibited this transport but, surprisingly, not the surface appearance of the short-chain sphingomyelin analog N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl(C6-NBD)-sphin-gomyelin as assayed by depletion of this lipid in the medium by the scavenger albumin. Transport of C6-NBD-sphingomyelin in the presence of brefeldin A was blocked by cyclosporin A and PSC 833, inhibitors of the multidrug resistance P-glycoprotein. The same was observed in HepG2 and HeLa cells, and for short-chain glucosylceramide, which demonstrates the general nature of the transporter-dependent sphingolipid translocation across the plasma membrane.
Golgi membrane skeleton: identification, localization and oligomerization of a 195 kDa ankyrin isoform associated with the Golgi complex
Authors: Beck Kenneth A. , Buchanan Jo Ann , Nelson W. James
Published: April 2021

To extend our finding of a Golgi-localized form of the membrane skeleton protein spectrin, we have identified an isoform of ankyrin that associates at steady state with the Golgi complex. Immuno-light and -electron microscopy show that this ankyrin isoform localizes to the perinuclear cytoplasm on tubular vesicular structures that co-stain with Golgi marker proteins. An antiserum raised against erythrocyte ankyrin, which was used to identify the Golgi ankyrin, recognized three prominent polypeptides of 220, 213 and 195 kDa in MDCK cells. Affinity purification of this antiserum against each of these MDCK cell ankyrins revealed that only an antibody specific for the 195 kDa form retained the ability to stain the Golgi complex; affinity purified antibody preparations specific for both the 220 and 213 kDa forms stained punctate and reticular cytoplasmic structures distinct from the Golgi complex. Antibody specific for the 195 kDa ankyrin did not recognize a recently identified 119 kDa ankyrin that is also localized to the Golgi. The 195 kDa Golgi ankyrin binds purified erythrocyte spectrin, and rapidly co-sediments with Golgi β-spectrin during brief, low speed centrifugation of Triton X-100 extracts of MDCK cells. Golgi ankyrin and β-spectrin are retained on tubular vesicular ‘Golgi ghosts’ following extraction of cultured cells with Triton X-100. Significantly, Golgi ghost tubules containing ankyrin/spectrin are co-linear with individual microtubules, suggesting a role for both Golgi membrane skeleton and microtubules in spatial localization of the Golgi. Golgi ankyrin dissociates from Golgi membranes during mitosis and in cells treated with brefeldin A, indicating that Golgi ankyrin has a dynamic assembly state similar to that of Golgi spectrin and other Golgi membrane coat proteins.
Cyclin dependent kinase 5, cdk5, is a positive regulator of myogenesis in mouse C2 cells
Authors: Lazaro Jean-Bernard , Kitzmann Magali , Poul Marie-Alix , Vandromme Marie , Lamb Ned J. C. , Fernandez Anne
Published: April 2021

We have examined the expression, activity and localization of cyclin dependent kinase 5 (cdk5), during myogenesis. Cdk5 protein was found expressed in adult mouse muscle. In murine C2 cells, both the protein level and kinase activity of cdk5 showed a marked increase during early myogenesis with a peak between 36 and 48 hours of differentiation, decreasing as myotubes fuse after 60 to 72 hours. This increase in cdk5 protein level was specific for differentiation and not simply related to cell cycle arrest since it was not observed in fibroblasts grown for 48 hours in low serum medium. Indirect immunofluorescence using mono-specific purified anti-cdk5 antibodies showed a low level cytoplasmic staining in proliferative myoblasts, a rapid increase in nuclear staining during the initial 12 hours of differentiation and a predominant nuclear staining in myotubes. Microinjection of plasmids encoding wild-type cdk5 into C2 myoblasts enhanced differentiation as assessed by both myogenin and troponin T expression after 48 hours of differentiation. In contrast, microinjection of plasmids encoding a dominant negative mutant of cdk5 inhibited the onset of differentiation. These data imply a previously unsuspected role for cdk5 protein kinase as a positive modulator of early myogenesis.
Vam3p, a new member of syntaxin related protein, is required for vacuolar assembly in the yeast Saccharomyces cerevisiae
Authors: Wada Yoh , Nakamura Norihiro , Ohsumi Yoshinori , Hirata Aiko
Published: April 2021

Syntaxins are thought to participate in the specific interactions between vesicles and acceptor membranes in intracellular protein trafficking. VAM3 of Saccharomyces cerevisiae encodes a 33 kDa protein (Vam3p) with a hydrophobic transmembrane segment at its C terminus. Vam3p has structural similarities to syntaxins of yeast, animal and plant cells. Δvam3 cells accumulated spherical structures of 200-600 nm in diameter, but lacked normal large vacuolar compartments. Loss of function of Vam3p resulted in inefficient processing of vacuolar proteins proteinase A, proteinase B and carboxypeptidase Y, and defective maturation of alkaline phosphatase. Subcellular fractionation and immunofluorescence microscopy showed that Vam3p was localized to the vacuolar membranes. Vam3p was accumulated in certain regions of the vacuolar membranes. We conclude from these observations that Vam3p is a novel member of syntaxin in the vacuoles and it provides the t-SNARE function in a late step of the vacuolar assembly.
Polarisation-dependent association of plectin with desmoplakin and the lateral submembrane skeleton in MDCK cells
Authors: Eger Andreas , Stockinger Andreas , Wiche Gerhard , Foisner Roland
Published: April 2021

The intermediate filament-binding protein plectin and cytokeratin were localised at the cellular periphery of fully polarised Madin-Darby canine kidney (MDCK) cells, whereas vimentin was primarily found in a perinuclear network. Confocal and immunoelectron microscopy revealed that plectin was restricted to areas underlying the lateral plasma membrane. It colocalised with fodrin, a component of the submembrane skeleton, and was closely associated with desmosomal plaque structures. Biochemically, plectin was shown to interact directly with immunoprecipitated desmoplakin in vitro. Upon loss of cell polarity in low calcium medium, plectin redistributed to a cytoplasmic vimentinand cytokeratin-related network, clearly distinct from diffusely distributed fodrin and internalised desmoplakin structures. The structural reorganisation of plectin was also reflected by an increased solubility of the protein in Triton X-100/high salt, and a decrease in its halflife from ∼20 to ∼5 hours. Furthermore, unlike cytokeratins and vimentin, desmoplakin and fodrin did not associate with plectin attached to magnetic beads in cell lysates of unpolarised cells, while all proteins formed a stable complex in polarised cells. Altogether, these data indicate that plectin is involved in the anchorage of intermediate filaments to desmosomes and to the submembrane skeleton in polarised MDCK cells.
When helicase and topoisomerase meet!
Authors: Duguet Michel
Published: April 2021

Several examples of direct interactions between helicases and topoisomerases have recently been described. The data suggest a possible cooperation between these enzymes in major DNA events such as the progression of a replication fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, and finally recombination, repair, and genomic stability. A first example is the finding of a strong interaction between T antigen and topoisomerase I in mammalian cells, that may trigger unwinding of the parental DNA strands at the replication forks of Simian Virus 40. A second example is the reverse gyrase from thermophilic prokaryotes, composed of a putative helicase domain, and a topoisomerase domain in the same polypeptide. This enzyme may be required to maintain genomic stability at high temperature. A third example is the finding of an interaction between type II topoisomerase and the helicase Sgs1 in yeast. This interaction possibly allows the faithful segregation of newly replicated chromosomes in eukaryotic cells. A fourth example is the interaction between the same helicase Sgs1 and topoisomerase III in yeast, that may control recombination level and genetic stability of repetitive sequences. Recently, in humans, mutations in genes similar to Sgs1 have been found to be responsible for Bloom’s and Werner’s syndromes. The cooperation between helicases and topoisomerases is likely to be extended to many aspects of DNA mechanisms including chromatin condensation/decondensation.
Cartilage associated protein (CASP) is a novel developmentally regulated chick embryo protein
Authors: Castagnola Patrizio , Gennari Massimo , Morello Roy , Tonachini Laura , Marin Oriano , Gaggero Alessia , Cancedda Ranieri
Published: April 2021

A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a strong signal was detected in the cartilage extracellular matrix of selected regions of the developing skeleton. Because of this localization of the antigen we named this protein cartilage associated protein (hereafter referred to as CASP).
Ligand recruitment by vinculin domains in transfected cells
Authors: Bubeck Peter , Pistor Susanne , Wehland Jürgen , Jockusch Brigitte M.
Published: April 2021

Vinculin, a prominent protein component of microfilamentmembrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge. In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system. To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes. When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes. In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis. A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little α-actinin and talin. The presence of only the vinculin head directed some of the fusion protein to focal contacts, and α-actinin recruitment was still ineffective. Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively. Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding. The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment. The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.
Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis
Authors: Walker Gary R. , Shuster Charles B. , Burgess David R.
Published: April 2021

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phos-

phorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycledependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.
Crystalline mitochondrial inclusion bodies isolated from creatine depleted rat soleus muscle
Authors: O’Gorman Eddie , Fuchs Karl-Hermann , Tittmann Peter , Gross Heinz , Wallimann Theo
Published: April 2021

Rats were fed a 2% guanidino propionic acid diet for up to 18 weeks to induce cellular creatine depletion by inhibition of creatine uptake by this creatine analogue. Ultrastructural analysis of creatine depleted tissues showed that mitochondrial intermembrane inclusion bodies appeared in all skeletal muscles analysed, after 11 weeks of feeding. Heart had relatively few even after 18 weeks of analogue feeding and none were evident in kidney, brain or liver. These structures were strongly immuno-positive for sarcomeric mitochondrial creatine kinase and upon removal from mitochondria, the inclusion bodies were shown to diffract to a resolution of 2.5 nm. Two-dimensional image analysis and three-dimensional reconstruction revealed arrays of creatine kinase octamers with additional components between the octameric structures. The same mitochondria had a 3-fold higher extractable specific creatine kinase activity than controls. Molecular mass gel filtration of inclusion body containing mitochondrial extracts from analogue fed rat solei revealed mitochondrial creatine kinase eluting as an aggregate of an apparent molecular mass ≥2,000 kDa. Mitochondrial creatine kinase of control soleus mitochondrial extract eluted as an octamer, with a molecular mass of 340 kDa. Respiration measurements of control solei mitochondria displayed creatine mediated stimulation of oxidative phosphorylation that was absent in analogue-fed rat solei mitochondria. The latter also had 19% and 14% slower rates of state 4 and maximal state 3 respiration, respectively, than control mitochondria. These results indicate that mitochondrial creatine kinase co-crystallises with another component within the inter membrane space of select mitochondria in creatine depleted skeletal muscle, and is inactive in situ.
Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein
Authors: Wacker Irene , Kaether Christoph , Krömer Andreas , Migala Andrea , Almers Wolf , Gerdes Hans-Hermann
Published: April 2021

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20°C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37°C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t1/2 of 20-30 minutes. Videomicroscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 μm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.
Concerted action of tenascin-C domains in cell adhesion, anti-adhesion and promotion of neurite outgrowth
Authors: Fischer Doris , Brown-Lüdi Marianne , Schulthess Therese , Chiquet-Ehrismann Ruth
Published: April 2021

We used a new approach to identify domains of chicken tenascin-C required for interaction with cells. Instead of expressing the parts of interest, we deleted them from an otherwise intact tenascin-C molecule and scored for the concomitant change in activity. As a starting point for all mutant constructs we expressed the smallest naturally occurring tenascin-C splice variant in vertebrate cells. The tenascin-C mutants had either deletions of all EGF-like repeats, all fibronectin type III repeats or of the fibrinogen globe. In double mutants the fibronectin type III repeats were deleted together with either the EGF-like repeats or the fibrinogen globe, respectively. All tenascin-C variants assembled correctly to hexameric molecules of the expected molecular characteristics. Intact tenascin-C and the mutant missing the fibrinogen globe did not promote adhesion of chick embryo fibroblasts, whereas both, the hexamers containing solely the fibrinogen globe or the EGF-like repeats were adhesive substrates and even supported cell spreading.

When tenascin-C was added to the medium of fibroblasts plated on fibronectin-coated wells, cell adhesion was blocked by intact tenascin-C, but not by mutants missing the fibrinogen globe. In neurite outgrowth assays using dorsal root ganglia, processes formed on all substrates except on the mutant missing only the fibrinogen globe, where the ganglia failed to adhere. The mutants missing the fibronectin type III repeats allowed more rapid neurite outgrowth than all other tenascin-C variants and the mutant consisting essentially of oligomerized EGF-like repeats was as active a substrate for neurite outgrowth as laminin. From the combined data, it is concluded that the activities of intact tenascin-C cannot be mimicked by investigating domain by domain, but the concerted action of several domains leads to the diverse cellular responses.
The mouse transcription factor Stat4 is expressed in haploid male germ cells and is present in the perinuclear theca of spermatozoa
Authors: Herrada Gilles , Wolgemuth Debra J.
Published: April 2021

STAT (signal transducer and activator of transcription) proteins have been shown to be essential transcription factors which mediate biological effects of cytokines. Although most of the STATs have been shown to be widely expressed, Stat4 mRNA has been detected in only a few tissues, including the testis. In the present study, immunoblot analysis confirmed that the presence of Stat4 protein was similarly restricted, with the highest level observed in testis. In situ hybridization, immunoblot, and immunohistochemistry analyses revealed that in the testis, Stat4 was abundantly and exclusively expressed in male germ cells which have completed meiosis, at the round and elongating spermatid stages. Cytolocalization at various times of spermatid differentiation showed that the level of Stat4 protein increased in parallel in both cytoplasm and nuclei. No specific nuclear translocation that would have been an indicator of Stat4 activation was observed at any stage of spermatogenic differentiation. Interestingly, the Stat4 transcription factor was localized to the condensing perinuclear theca of spermatids, a localization that was confirmed by selective biochemical extraction of thecal proteins. Since the theca is known to depolymerize in the cytoplasm of the oocyte during the hours following fertilization, we hypothesized that sperm Stat4 would represent an original paternal contribution to the fertilized egg which may be involved in the onset of zygotic transcription.
Involvement of the rfp tripartite motif in protein-protein interactions and subcellular distribution
Authors: Cao Tongyu , Borden Kathrine L. B. , Freemont Paul S. , Etkin Laurence D.
Published: April 2021

The ret finger protein (rfp) is a member of the B box zinc finger gene family which possesses a tripartite motif consisting of a RING finger, B box finger, and a coiled-coil domain. Rfp is expressed at specific stages of spermatogenesis and in various adult mouse and human tissues. It becomes oncogenic when the tripartite domain is recombined with the tyrosine kinase domain of the ret protooncogene. Many of the B box family proteins function as homodimers, although the role of the individual components of the tripartite motif in this process remains unclear. We demonstrate that rfp homomultimerization occurs through the coiled-coil domains; however, while the B box is not an interacting interface itself, its structural integrity is necessary for this interaction to occur. This is the first evidence that the B box zinc finger domain is involved in regulating protein-protein interactions. Interestingly, we find that mutations of the RING finger and B box affect the subcellular compartmentalization of rfp in various cell lines. These results demonstrate that the interactions of rfp with itself and its association with specific subcellular compartments is dependent upon the function of all of the components of the tripartite motif. It is likely that these domains play a crucial role in the function of the rfp protein in normal cell differentiation and in its transformation potential in the recombined state.