bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation, distribution and dissociation of intercellular junctions in the lens
Authors: Gruijters W. T. M. , Kistler J. , Bullivant S.
Published: April 2021

A 70000 Mr membrane protein (MP70) has previously been identified as a specific component of lens intercellular junctions. In this paper we use anti-MP70 immunofluorescence microscopy of dissected fibre bundles to study the formation, distribution and dissociation of junctional plaques in the outer cortex region of the sheep lens. Abundant, small junctional plaques are assembled de novo in the broad sides of the elongating fibres near the equatorial lens periphery. In fully elongated, pole-to-pole fibres, junctional plaques are generally larger, and while dispersed on the broad sides of the fibres in the equatorial lens plane, these junctions line up in the middle of the broad and narrow sides of the fibres in the lens polar regions. This precisely defined positioning is independent of junction size and hence cannot solely be explained by the constraints of fibre width. Junctional plaques fragment to smaller sizes and MP70 is cleaved to MP38 in mature, enucleated fibres located in the deeper portions of the lens outer cortex. These results demonstrate a dynamic aspect of lens intercellular junctions and show that they are positioned in a precise fashion, possibly in association with other membrane or cytoskeletal components.
Movements of fluorescent probes in the mechanism of cell fusion induced by poly(ethylene glycol)
Authors: Ahkong Quet F. , Desmazes Jean-Pierre , Georgescauld Dinu , Lucy Jack A.
Published: April 2021

It has been claimed that purified poly(ethylene glycol) (PEG) is able only to aggregate cells and not to fuse them. In our hands, purified PEG 6000 (recrystallized/dialysed) induces both aggregation and fusion of human erythrocytes, and the mechanism of fusion by the purified polymer has been investigated with fluorescent probes. No movement of a carbocyanine probe or of octadecyl rhodamine B chloride from labelled to unlabelled cells occurred in the absence of PEG or with cells treated with concanavalin A, protamine or spermine. With 40 % PEG, however, both probes immediately started to diffuse into the membranes of unlabelled cells. This indicates that continuity between the phospholipid bilayer membranes of adjacent erythrocytes (i.e. membrane fusion) is established within seconds in concentrated solutions of the polymer, and precedes the cell fusion event that is induced by purified PEG. These observations are consistent with the idea that micro-regions of shared phospholipid bilayer may be formed in the membranes of cells when they are forced together as a consequence of the dehydrating action of PEG. Intact erythrocytes were cytoplasmically labelled with 6-carboxyfluorescein to avoid the possibility that loading the cells with a cytoplasmic marker by hypotonic haemolysis might modify their response to PEG. Unlike the lipid probes, carboxyfluorescein did not diffuse from labelled to unlabelled cells in the presence of 40% PEG, and there was little diffusion on subsequent dilution of the polymer solution to 13%. However, after the PEG solution had been replaced by an isotonic buffer, a rapid transfer of the cytoplasmic fluorophore to unlabelled cells often occurred. This is considered to be more consistent with the osmotic rupture of a membranous barrier, such as a shared bilayer, between the labelled and unlabelled cells than with the return of cytoplasmic viscosity to normal when the PEG is removed. Possible reasons are discussed for the reported inability of purified PEG to fuse fibroblasts with hypotonically loaded human erythrocytes.
Evidence for heterogeneity in the 160/165×103mr glycoprotein components of desmosomes
Authors: Jones Jonathan C. R. , Vikstrom Karen L. , Goldman Robert D.
Published: April 2021

We have prepared both monoclonal and polyclonal antibody preparations directed against the 160/165 ×103Mr glycoproteins (desmogleins) of bovine tongue epithelial desmosomes. The polyclonal antibody preparation recognizes desmosomes in a number of mouse tissues, e.g. mouse skin, heart, bladder and trachea, as determined by immunofluorescence microscopy. Furthermore, the polyclonal antibodies recognize poly-peptide(s), present in the high salt, Triton-insoluble residues (‘cytoskeleton preparations’) of mouse skin, heart, bladder and trachea, which comigrate with the 160/165×103Mr glycoproteins of bovine tongue epithelial desmosomes as determined by ‘Western’ immunoblotting. Conversely, the monoclonal 160/165 × 103Mr antibody preparation recognizes desmosomes of stratified squamous epithelial tissues but not desmosomes in other tissue types. Moreover, whereas the monoclonal antibodies recognize 160/165 × 103Mr polypeptides in mouse skin cell cytoskeletons they show no immunoreactivity with the cytoskeleton preparations of mouse bladder, trachea and heart following immunoblotting. These results suggest therefore that although there are conserved epitopes of the 160/165 × 103Mr glycoproteins there are also epitopes of these molecules which vary from tissue to tissue.

Double label immunofluorescence observations of cryostat sections of mouse skin using the monoclonal antibodies and antibodies directed against desmoplakin, a plaque component of desmosomes, reveal that the monoclonal antibodies do not recognize certain desmosomes in basal cells which are recognized by desmoplakin antibodies. Indeed, double label observations of cryostat sections of mouse skin using the monoclonal antibodies and human autoantibodies which react with hemidesmosomal components suggest that the monoclonal antibodies stain desmosomes located along the apical surfaces of basal cells but fail to recognize desmosomes along the lateral surfaces of these same cells. The latter desmosomes, however, are recognized by the polyclonal 160/165 × 103Mr antibody preparation. We discuss the possibility that the inherent polarity of basal epithelial cells is manifested in modifications of the 160/165×103Mr glycoproteins in desmosomes located along different surfaces of basal cells.
Glass beads load macromolecules into living cells
Authors: Mcneil Paul L. , Warder Elizabeth
Published: April 2021

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75-500 μm diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10000Mr dextran; and 79% with a 150 000Mr dextran using beadloading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.
The gap junction
Authors: Warner Anne
Published: April 2021

Gap junctions are intercellular structures that link cells and allow them to exchange ions and small metabolites without recourse to the extracellular space (for reviews and early references, see Goodenough, 1979; Loewen-stein, 1981). They are found in every organ including the nervous system, where they form electrical syn-apses; the only exception being fully differentiated skeletal muscle. Gap-junctional communication is present between all cells in early embryos, regardless of eventual developmental fate, at times when cellular interactions are known to be taking place (Potter et al. 1966). The widespread occurrence of gap junctions has led to the proposal that they play an important part in coordinating cellular signals, including those involved in growth control and embryogenesis.
Cytolocalization of prosomes as a function of differentiation
Authors: De Sa M.F. Grossi , De Sa C. Martins , Harper F. , Coux O. , Akhayat O. , Pal J. K. , Florentin Y. , Scherrer K.
Published: April 2021

Prosomes, ubiquitous ribonucleoprotein (RNP) particles of defined biochemical and morphological structure, first isolated as a subcomplex of the repressed globin mRNP in avian and mouse erythroblasts, were also found in the cytoplasm of other vertebrates associated with other mRNAs. Here we show that prosomes are also present in the cell nucleus and, furthermore, that the cytolocalization of specific prosomal peptides is a function of differentiation. Four monoclonal antibodies, raised against the duck prosomal proteins, p27K, p28K, p29K and p31K (K = 103Mr) react to variable degree with prosomes of chicken, mouse, and human cells. Immunocytochemical and biochemical analyses show that all four antigens are present in both the cytoplasm and the nucleus of avian erythroblasts and avian erythroblastosis virus (AEV)-transformed erythroleukaemic cells. Interestingly, the prosomes disappear in the course of the terminal differentiation of erythroblasts to mature erythrocytes. Although all the four prosomal antigens tested are present in both the nuclear and cytoplasmic compartments, slight differences in the immunofluorescent patterns indicate that each antigen may have a particular cytological distribution that varies in the course of differentiation.
Culture patterns and sorting of rat Sertoli cell secretory proteins
Authors: Ueda Hiroshi , Tres Laura L. , Kierszenbaum Abraham L.
Published: April 2021

A cocultivation chamber and two types of permeable substrates have been used to study: (1) the culture patterns of rat Sertoli and peritubular cells, and Sertoli cells cocultured with spermatogenic cells or peritubular cells; and (2) the polarized secretion of Sertoli cell-specific proteins transferrin, S70 and S45-S35 heterodimeric protein. Substrates included a nylon mesh (with openings of 100 μm) coated with extracellular matrix (ECM) material and an uncoated microporous filter (with pores of O·45 μm). Sertoli cells cultured on ECM-coated nylon mesh organized a continuous sheet of multilayered epithelial cells essentially devoid of spermatogenic cells while peritubular cells formed a layer of squamous cells. Sertoli cells cultured on uncoated microporous substrate formed a continuous sheet of cuboidal epithelial cells with numerous basal cytoplasmic processes projecting into the substrate and abundant apically located spermatogenic cells, while peritubular cells organized one or two layers of loose squamous cells, [35S]methionine-labelled secretory proteins resolved by two-dimensional polyacrylamide gel electrophoresis and autoradiography displayed cell-specific patterns that were slightly influenced by the type of substrate. Sertoli cells cocultured with peritubular cells on uncoated microporous substrate under conditions that enabled separation of apical and basal surfaces, secreted proteins in a polarized fashion. While transferrin was released bidirectionally, S45 – S35 heterodimeric protein was released apically. S70 was detected in both apical and basal compartments. We conclude from these studies that: (1) the number of spermatogenic cells decreases when Sertoli-spermatogenic cell cocultures are prepared on ECM-coated nylon substrate; and (2) Sertoli cells in coculture with spermatogenic or peritubular cells on uncoated microporous substrate, organize continuous sheets displaying polarized protein secretion.
Evidence for septate junctions in the syzygy of the protozoon Gregarina polymorpha (Protozoa, Apicomplexa)
Authors: Dallai Romano , Talluri Maria Vegni
Published: April 2021

A septate junction is described in reproductive pairs of the protozoon Gregarina polymorpha, using conventional thin sections, lanthanum tracer and freeze-fracture techniques. The septate junction is established between the plasma membranes at the tips of the joined epicytic folds. It is characterized by an intercellular space of 14–17 nm traversed by septa with a repeat of 15–25 nm. Lanthanum-treated material exhibits transparent curves forming a meshwork. Freeze fracture replicas show membrane modifications in the shape of short rows of intramembranous particles on the E fracture face of the plasma membrane. The significance of the finding of such a septate junction between protozoan cells is discussed.
Supercoils in plant DNA: Nucleoid sedimentation studies
Authors: Stoilov L. M. , Zlatanova J. S. , Vassileva A. P. , Ivanchenko M. G. , Krachmarov Ch. P. , Genchev D. D.
Published: April 2021

Plant nuclei have been studied with respect to the three-dimensional structure of DNA. Nucleoids derived from nuclei by non-ionic detergent and high salt treatment were analysed by sedimentation in a series of sucrose gradients containing increasing amounts of the intercalating agent ethidium bromide. In addition the nucleoid sedimentation behaviour was investigated following gamma irradiation. The results show that plant DNA is supercoiled, as is the DNA from the other eukaryotes studied, and contains approximately the same concentration of superhelical turns but probably relatively fewer DNA superhelical loops. The plant nuclear populations in all cases studied give rise to two distinct nucleoid bands. These have been characterized by electron microscopy and by their DNA and protein content. The possible origin of the two bands is discussed.
Shedding of cytoplasmic actins by developing muscle cells
Authors: Denning G. M. , Kim I. S. , Fulton A. B.
Published: April 2021

Young myotubes develop spots or macules of actin and alpha actinin under and adjacent to slender actin strands. Macules are found only before sarcomere formation. They contain cytoplasmic actin, small amounts of alpha actin, and alpha actinin but are devoid of tubulin, myosin and vimentin. In the electron microscope, they are seen to contain small filaments but no other organelles. Macules are found on the culture dish and in the culture medium, as well as on the lower and lateral surfaces of the cells. Both emetine and cycloheximide, at doses that inhibit protein synthesis, accelerate formation and shedding of macules. This presents the first observation of a cellular system in which the changeover from a generalized cytoskeleton to a tissue-specific cytoskeleton involves shedding of the cytoplasmic isoforms of cytoskeletal proteins.
Microtubules associate with actin-containing filaments at discrete sites along the ventral surface of Allogromia reticulopods
Authors: Bowser Samuel S. , Travis Jeffrey L. , Rieder Conly L.
Published: April 2021

We have investigated the distribution of actin and microtubules in pseudopodial networks (reticulopods) of the protozoan Allogromia sp., strain NF, in order to help elucidate the respective roles these components play in network organization and motility. Double-label fluorescence studies with tubulin antibodies and tetramethyl-rhoda-mine (TMR)-phalloidin reveal that microtubules and filamentous actin co-localize in regions where trunk pseudopods contact the substratum and splay to form the pseudopodial network; distal to these regions the network contains numerous microtubules but little or no F-actin. Similar results were obtained using various commercial actin antibodies. Correlative anti-actin immunofluorescence and high-voltage electron microscopy of serial 0-25 jtm sections reveal that actin is contained within discrete electronopaque, fan-shaped structures distributed along the cytoplasmic aspect of the ventral reticulopo-dial membrane. Electron microscopy of serial 100 nm sections from conventionally fixed specimens confirms that these actin-rich plaques are composed of a felt of roughly parallel, 5nm diameter filaments. A subset of parallel and often bundled microtubules is enmeshed within, or contacts the periphery of, these filament plaques. Upon leaving a plaque, bundled microtubules frequently splay into smaller bundles. These observations are consistent with the hypothesis that interactions between microtubules and actin-containing microfilaments, particularly at substratum adhesion points, are involved in various aspects of reticulopodial motility, particularly network morphogenesis and cell body locomotion.
Effects of elevated intracellular magnesium on cytoskeletal integrity
Authors: Prescott A. R. , Comerford J. G. , Magrath R. , Lamb N. J. C. , Warn R. M.
Published: April 2021

Increasing the intracellular magnesium concentration of PtK2 cells by 1 mM or more resulted in the disassembly of the interphase microtubule array over a period of 5 min after microinjection. This effect was found to be both transient and fully reversible, with the microtubule arrays reforming after further incubation. These effects were studied using immunofluorescence microscopy of fixed cells, and also in living cells using rhodamine-tubulin or rhodamine-conjugated anti-tubulin antibodies and image intensification and enhancement techniques.

Simultaneously and accompanying the disassembly of the microtubule arrays the F-actin stress fibres also disappeared, usually leaving the peripheral and perinuclear F-actin microfilaments intact. In contrast, increasing intracellular magnesium appeared to have no effect on the vimentin-containing intermediate filaments of PtK2 cells.

These effects on the cytoskeleton were specific to magnesium and could not be mimicked by either microinjection of injection buffer of equivalent ionic strength or sham injection. Raising the intracellular free calcium to the same extent resulted in the disassembly of the microtubule network, but appeared to have no effect on the Factin stress fibres.
Estramustine binds map-2 to inhibit microtubule assembly in vitro
Authors: Stearns Mark E. , Tew Kenneth D.
Published: April 2021

We have investigated the ability of estramustine to bind to rat brain microtubule-associated proteins (MAPs) and purified MAP-2 in vitro. [3H]estramustine’s relative affinity for tubulin and MAPs was assessed by gel filtration chromatography, immunoprecipitation and binding assays. Scatchard analysis demonstrated a specific affinity of the drug for MAP-2. Calculations from kinetic parameters and non-linear regression analysis gave a Kd of 15 μM, and a Bmax of 3·4×10−7 M ml−1. Extrapolation of this value suggested that each MAP-2 molecule binds approximately 20 molecules of estramustine.

Microtubule assembly studies and SDS-poly-acrylamide gel electrophoresis revealed that at 20–60 μM levels, estramustine inhibited the association of MAPs with taxol microtubules. Turbidity (Asso) studies further demonstrated that 20–60μM estramustine inhibited MAP-2-driven tubulin assembly and produced microtubule disassembly. Electron-microscopic studies confirmed the centrifugation and turbidity results. The data demonstrated that estramustine can bind MAPs and MAP-2 specifically, thereby inhibiting microtubule assembly.
The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe
Authors: Hagan Iain M. , Hyams Jeremy S.
Published: April 2021

We have characterized the changes in microtubule organization that occur through the cell division cycle of the fission yeast Scbizosaccbaro-tnyces pombe by indirect immunofluorescence microscopy. During interphase, groups of cytoplasmic microtubules, independent of the spindle pole body (SPB), form an array extending between the cell tips. These microtubules are involved in positioning the nucleus at the cell equator and in the establishment of cell polarity. At mitosis, the interphase array disappears and is replaced by an intranuclear spindle extending between the now duplicated SPBs. Elongation of the spindle sees the appearance of astral microtubules emanating from the cytoplasmic face of the SPBs. These persist until the end of anaphase whereupon the spindle microtubules depolymerize and two microtubule organizing centres (MTOCs) at the cell equator re-establish the interphase array. We have used the unique properties of various cell division cycle mutants to investigate further the function of these different microtubule arrays and their temporal and positional control.
Regulation of cell shape in Euglena gracilis v. time-dependent responses to ca2+ agonists and antagonists
Authors: Lonergan Thomas A. , Williamson Lura C.
Published: April 2021

The daily changes in cellular shape observed in growth-synchronized cultures of Euglena gracilis Klebs strain Z, were altered by exposure to Ca2+ channel agonists and antagonists. The response of the cells to these pharmacological agents depended, in part, on the time in the growth cycle that the cells were exposed. The Ca2+ channel blockers verapamil and nifedipine and the intracellular Ca2+ antagonist TMB-8 all caused cell rounding when elongated cells from the middle of the light cycle were treated. These results were the same as with other methods used to deprive cells of extracellular Ca2+, such as exposure to EGTA or resuspension in Ca2+-free medium. The cell response in mid light cycle to the channel blockers was reversible by simultaneous exposure to CaCl2, and the nifedipine response was also reversed by simultaneous exposure to the structurally related Ca2+ agonist BAY-K 8644. Exposure of cells in the first hour of the light cycleto verapamil, nifedipine or TMB-8 caused an unexpected result. Instead of preventing the round cells from elongating in the first portion of the light cycle, as do LaCl3, EGTA or resuspension in Ca2+-free medium, a greater than expected percentage of elongated cells was found in the treated population. This represents the first instance in which the biological clock control over the rate and extent of cell elongation was accelerated. The calcium agonist CGP-28392 did not have an effect on cell elongation in the early portion of the light cycle but caused cell rounding in the middle of the light cycle. The calcium agonist BAY-K 8644 did not cause any shape changes alone, but was capable of reversing the effects of nifedipine in the middle of the light cycle.
Elongation of g1 phase by transient exposure of rat 3y1 fibroblasts to caffeine during the previous and present generations
Authors: Okuda Atsuyuki , Kimura Genki
Published: April 2021

When density-arrested rat 3Y1 fibroblasts were stimulated to enter S phase by seeding sparsely in fresh medium, caffeine inhibited this stimuΛlation. When compared at the doses that gave the same levels of the inhibition of entry into S phase, caffeine inhibited protein synthesis to a far lesser extent than cycloheximide. This indicates that caffeine affects some event(s) specific to entry into S phase rather than general protein synthesis. When cells synchronized at early S phase were exposed to caffeine, progression of S and G2 phases was prolonged by only 1 h (from 6 h to 7 h). However, after removal of caffeine at mitosis, the G1 phase was prolonged for 5 h (from 11 h to 16h). These results are consistent with our model that the initiation of S phase is regulated throughout the period between the adjacent S phases. When cells were incubated with normal medium conΛtaining serum during the S and G2 periods, a subsequent 6-h pulse exposure to caffeine caused prolongation of G1 phase for 7h (from 11 h to 18 h). On the other hand, when cells were incuΛbated in the absence of serum during these periods, the prolongation was only 2h (from 16 h to 18 h). Similarly, entry into S phase was proΛlonged only 2h, when a 6-h pulse exposure to caffeine was given immediately after release from density arrest or serum-deprivation arrest. These results indicate the involvement of the relaxation process, which is not affected by cafΛfeine, when serum-deprived cells or density-arΛrested cells restore the process prerequisite for entry into S phase.
Characterization of a spore protein inducing factor from Dictyostelium discoideum
Authors: Gibson F. P. , Hames B. David
Published: April 2021

Spore coat protein synthesis during development by submerged pseudoplasmodia of Dictyostelium discoideum requires a low molecular weight factor secreted by cells incubated at high density in buffer. The further characterization of this spore protein inducing factor (SPLF) is reported. Its behaviour during anion-exchange chromatography and the loss of activity upon esterification suggests the presence of a carboxylic acid group essential for biological activity. Gel permeation chromatography resolves a major SPIF activity with Mr ∼ 160-200 and a minor activity with Mr∼ 340-420. Anion-exchange HPLC further resolves the major SPIF activity into four components, one major and three minor. Methionine, analogues of methionine, and precursors of methionine biosynthesis are all effective in maintaining spore coat protein synthesis. Conditioned medium contains methionine at a concentration sufficient to account for its SPIF activity and this activity is abolished by cyanogen bromide treatment. These results indicate that SPIF is either methionine or a close analogue of methionine.
The behavioural repertoire of neutrophils requires multiple signal transduction pathways
Authors: Lackie J. M.
Published: April 2021

Signal-response transduction is usually investigated in cells that produce an easily assayed and straightforward response to a well-defined stimulus; the repertoire of the cell is often rather uninteresting. An enjoyable feature of working with neutrophil leucocytes is that their behaviour is far from simple, although at times this can be a mixed blessing. To carry out their primary defensive function in vivo, neutrophils must go through a complex sequence of activities, in the correct order, and in the right places. In an acute inflammatory response neutrophils adhere to, and move over, the luminal surface of endothelial cells, penetrate the endothelium, and move through connective tissue towards the site of damage. Having arrived, and stopped moving, neutrophils then engage in phagocytosis or secrete hydrolytic enzymes to digest large foreign objects (Lackie, 1982). The early stages require changes in adhesive and motor capacity, the late stages of the process are marked by a dramatic activation of the metabolism of the cell. Activities that are essential in the late stages would be inappropriate, and destructive of normal tissue, in the early stages.
Characterization of mutant human fibroblast cultures transformed with simian virus 40
Authors: Miranda Armand F. , Duigou Gregory J. , Hernandez Evelyn , Fisher Paul B.
Published: April 2021

Fibroblast cell strains derived from a normal individual and from eight patients with various genetic mutations were transformed by a smallplaque variant of simian virus 40 (SV40, strain 776), cloned and studied after long-term in vitro maintenance. Seven of the cultures continued to express the mutant phenotype. Cultures derived from a patient with phosphoglycerate kinase I deficiency exhibited reappearance of normal enzyme activity after transformation. Compared to untransformed controls, all transformed cultures displayed decreased population doubling times, an increase in the relative number of cycling cells and increased saturation density on solid substrates, and did not show evidence of cellular senescence after long-term cultivation. Unlike previous studies on wild-type SV40-transformed human fibroblasts, the majority of cultures transformed by the small-plaque variant of SV40 did not exhibit signs of crisis. The cells also exhibited a decreased dependence on serum and were able to grow in semi-solid medium. The different transformed cultures expressed variable levels of SV40 large T-antigen, synthesized some infectious SV40 virus, and contained both unique arrangements and quantities of covalently integrated and episomal SV40 DNA. No correlation was observed between the rate of growth and synthesis of infectious virus in the different transformed clones. These studies indicate that this small-plaque variant of SV40 can be used effectively to generate long-lived human cultures, which generally retain their mutant phenotype. Transformation with this SV40 variant permits the generation of large quantities of clonal cell cultures for the biochemical and molecular analysis of their genetic defects.
Microtubules and microfilaments in tip growth: Evidence that microtubules impose polarity on protonemal growth in Physcomitrella patens
Authors: Doonan J. H. , Cove D. J. , Lloyd C. W.
Published: April 2021

In this study we compare the contributions of Factin and microtubules to tip growth in filamentous cells of the moss Physcomitrella patens.

In tip growth, expansion seems to be restricted to the hemispherical apical dome. Cytoskeletal elements have been suspected, from drug studies, to be involved in this but electron microscopy has generally not confirmed the presence of an apical cytoskeleton. However, in a previous immunofluorescence study we reported that microtubules could be seen to focus upon the apical dome in tip cells of the moss P. patens. In the present investigation F-actin has also been detected at the apices of these cells. Anti-cytoskeletal drugs were therefore used to differentiate between the roles of actin filaments and microtubules in tip growth.

At high concentrations (30 μM), the herbicide cremart de-polymerized microtubules and caused tip swelling. F-actin was still present under such conditions but its fragmentation by cytochalasin D suppressed this herbicide-induced swelling. On its own, cytochalasin D arrested tip growth without causing swollen tips.

At lower concentrations, cremart disorganized microtubules rather than causing their complete depolymerization. Under these conditions, new but swollen growing points were initiated along the filament. The addition of taxol to cremart-treated filaments tended to reduce swelling and to re-polarize outgrowth. With particular combinations of these drugs, multiple lateral outgrowths were initiated in the vicinity of the nucleus.

It is concluded: (1) that F-actin is present at the tips of Physcomitrella caulonemal apical cells; (2) that unfragmented F-actin is necessary for outgrowth; (3) that even disorganized microtubules permit some degree of outgrowth but that an unperturbed distribution of axial microtubules, focussing upon an apex, is essential in order to impose tubular shape and directionality upon expansion.
The initiation, maintenance and termination of dna synthesis: a study of nuclear dna replication using Amoeba proteus as a cell model
Authors: Ord M. J.
Published: April 2021

By means of the nuclear transfer technique for amoeba, combinations of nuclei and cytoplasms from all parts of the cell cycle were available for examining the individual roles of the nucleus and cytoplasm in nuclear DNA replication. Neither 5-phase nor division sphere cyto-plasm proved capable of initiating a new round of nuclear DNA synthesis in the G2 nucleus. There was some indication that G2 nuclei which were transferred into early prophase cells, i.e. before the formation of a regular division sphere, did incorporate more [3H]thymidine than control G, nuclei.

Positive proof of the induction of DNA synthesis in ‘immature’ nuclei was observed in only two cases. When young G2 nuclei were transplanted into late G2 amoebae, the addition of the donor nucleus generally resulted in the older nucleus being held in a late G2 phase until the younger nucleus passed through its G2. Division of 90 % of heterophasic homokaryons was synchronous, with a subsequent synchrony of DNA synthesis.

A study of variance in [3H]thymidine incorporation by S nuclei sharing the same cytoplasm – using binucleate, trinucleate and multinucleate homokaryons — showed that nuclei through the peak-5 period synthesized DNA at approximately similar rates. The large differences in PHjthymidine incorporation by nuclei of amoebae of equal age appear due to differences in endogenous precursor pools. These would vary both with differences in food intake and with the draining of remote precursor pools for simultaneous cellular activities, particularly RNA synthesis. When sharing the same cytoplasm nuclei in peak 5 incorporated similar amounts of [3H]thymidine. Though cytoplasm did not influence the progress of DNA replication by a nucleus, it did influence the use of exogenous [3H]thymidine by the cell, and in so doing caused much of the variation observed in the labelling of nuclei during S. Nuclei sharing the same cytoplasm, and so subject to the same precursor pool changes, incorporated similar amounts of exogenous thymidine.

Once DNA synthesis had been initiated it continued to completion regardless of the cyto-plasm which surrounded it. Thus neither the maintenance nor termination of DNA synthesis required a special cytoplasmic state.
Faults in the square lattice of mammalian skeletal muscle z-disks
Authors: Rowe R. W. D. , Morton D. J.
Published: April 2021

The ordered square lattices of filaments in the Z-disk and juxtaposed I-band regions proposed by current models of Z-disk structure were compared with those observed by electron microscopy. It was commonly found that there were faults in the lattices of both the Z-disk and I-band regions. Attention is drawn to these faults and the consequences of their presence are discussed in relation to their influence on myofibril size and proliferation.
Pollen-wall proteins: the fate of intine-held antigens on the stigma in compatible and incompatible pollinations of Phalaris tuberosa l.
Authors: Knox R. B. , Heslop-Harrison J.
Published: April 2021

By the use of an immunofluorescence technique, the main source of the antigens released by the pollen grains of Phalaris tuberosa L. (Gramineae) on leaching has been shown to be the intine. The main concentration is in the thickened zone underlying the germination pore. The intine is also the site of various hydrolytic enzymes.

The fate of the intine-held antigens has been followed in compatible and incompatible pollinations. They are lost on to the stigma within 5 – 10 min, whether or not the pollen grains germinate. Where germination does occur after a compatible pollination, the antigens remain spread on the surface of the stigma cells after the tubes have penetrated. There is no indication that antigenic material of the same type is released during the further growth of the tubes.

The possible roles of the intine-held materials as recognition substances in inter- and intra-specific compatibility reactions are discussed.
The neuropil of the glomeruli of the olfactory bulb
Authors: Pinching A. J. , Powell T. P. S.
Published: April 2021

The neuropil of the glomeruli of the rat olfactory bulb has been studied with the electron microscope with a view to elucidating the type of processes involved - dendrites, appendages and axons - their cellular identity, and the synaptic relationships they establish. The problems encountered in defining these are considered and criteria based on the previous study of neuron types and on examination of serial sections are put forward. The glomeruli are large structures containing many thousands of processes and are the sole site of termination of the olfactory receptor axons. The terminals of the latter are characteristically electron-dense, allowing identification in normal material; they run through the glomeruli making many synapses by means of spherical vesicles and asymmetrical thickenings on to all types of dendritic profile. The glomerular dendritic arborizations of mitral and tufted cells, which are indistinguishable from each other, start as large, fairly regular, pale profiles but become increasingly varicose as they branch and diminish in size. They regularly show groups of spherical vesicles, often in association with asymmetrical synaptic thickenings directed from the dendrite; these are typically associated with return, reciprocal, synapses of the symmetrical type from profiles containing large flattened vesicles. These latter profiles are those of the dendrites and gemmules of periglomerular cells; the dendrites are of irregular outline and give rise to many appendages, mostly gemmules making synaptic contact with mitral or tufted cell dendrites. A small number of pale axon terminals containing either small or large flattened vesicles, derived from shortaxon and periglomerular cells respectively, synapse with symmetrical thickenings on to the periglomerular cell dendritic processes. Close associations of particular types of axo-dendritic and dendro-dendritic synapses on interconnecting processes, termed synaptic patterns, are described and their significance considered. The nature of the glomerular interactions is discussed and then placed in the context of other, smaller glomeruli in the central nervous system; certain common principles of glomeruli are suggested.
The intracellular origin of flagellar hairs in the dinoflagellate Woloszynskia micra leadbeater & dodge
Authors: Leadbeater B. S. C.
Published: April 2021

The arrangement of fine hairs on the longitudinal and transverse flagella of Woloszynskia micra is illustrated and discussed. Dilations of the nuclear envelope and endoplasmic reticulum in W. micra, similar in appearance and location to those containing flagellar hairs in other algal and fungal zoids, are found to contain bundles of fibrils. These fibrils are approximately the same width and length as the flagellar hairs. For the above reasons it is now considered that the flagellar hairs in the dinoflagellate W. micra are formed intracellularly within dilations of the nuclear envelope and endoplasmic reticulum and later deposited on the flagella.
C-microtubules in isolated mitotic spindles
Authors: Cohen W. D. , Gottlieb T.
Published: April 2021

Microtubules with incomplete cylindrical structure are present in isolated mitotic spindles of the sea urchin, Arbacia punctulata. In cross-section they appear C-shaped, and are thus similar to the ‘C-microtubules ‘or ‘C-filaments’ observed previously in other systems. The C-micro-tubules are not uniformly distributed within isolated spindles, but are typically numerous in the interzonal region of anaphase spindles and in the metaphase chromosome ‘plate’. In chromosome-to-pole regions they are seen much less frequently, and microtubules with the usual O-configuration predominate. Counts of C- and O-microtubules in anaphase spindle crosssections of known location show an inverse relationship between the number of C-microtubules present and the total number of microtubules present. The observations suggest that the C-microtubules are not simple artifacts of fixation or isolation, but rather may represent a stage of microtubule disassembly which occurs in the interzone during isolation or during anaphase in vivo. The alternate possibility of assembly is not excluded, however. The significance of C-microtubules is further discussed with respect to their occurrence in other systems, and to potential differences between mitotic microtubules.
Dna from isolated pellicles of Tetrahymena
Authors: Flavell R. A. , Jones I. G.
Published: April 2021

Large pellicle fragments were isolated from Tetrahymena pyriformis, strain T, by 2 procedures: homogenization after treatment with 40 % ethanol at − 20 °C, or direct homogenization in a sucrose-EDTA buffer. All preparations contained entrapped mitochondria. DNA prepared from these pellicles was analysed on a CsCl gradient, and contained 3 components of buoyant densities 1·685, 1 ·688, and 1698 g cm−3 in variable proportions. The component at 1685 g cm−3 is similar in density to mitochondrial DNA and those at 1·688 and 1·698 g cm−3 to components of nuclear DNA. Most pellicle preparations contained a higher proportion of the heavy component (1·698 g cm−3) than does nuclear DNA. A similar enrichment of this component could be demonstrated in high-speed pellets from fragmented nuclei. No unique pellicle-associated component could be demonstrated. No DNA could be isolated from very pure preparations of oral plates and we conclude that there is no evidence for the presence of a specific DNA associated with the pellicle.
A comparative study of three methods for the ultrastructural demonstration of glycogen in thin sections
Authors: Vye M. V. , Fischman D. A.
Published: April 2021

In order to evaluate 3 staining methods for demonstration of glycogen in thin sections, 2 tissues containing an abundance of this carbohydrate in β-particle form were studied. Tissues were aldehyde-fixed, postfixed in osmium tetroxide, embedded in Araldite and sectioned in the usual manner without special precautions. The first method for staining thin sections employed a sequential combination of periodic acid, thiosemicarbazide and osmium tetroxide vapour, while in the second procedure a silver protein solution was substituted for the osmium tetroxide vapour. The third technique utilized periodic acid, sodium chlorite and uranyl acetate, also in sequential combination. Each method yielded glycogen particles of greater electron density than were seen in sections stained by the usual uranyl acetate-lead citrate procedure. Under high magnification, considerable method-dependent variation in the appearance of the glycogen granules was noted. Particulate substructure, only faintly visible in routinely stained sections, was easily resolved with the periodic acid-thiosemicarbazide-silver protein technique. Conversely, periodic acid-thiosemicarbazide—osmium tetroxide completely obscured this substructure. With periodic acid—sodium chlorite—uranyl acetate, glycogen particles appeared larger, more confluent, and of a less regular outline than with the other methods. Sections were also stained by incubation in periodic acid prior to treatment with lead citrate. The alteration in appearance of the glycogen granules produced by this modification was so great that high-resolution analysis of particle size and substructure could not be undertaken. The usefulness of the procedures investigated here resides in their ability to stain glycogen in thin sections in an intense and selective manner.
Evidence that cyclic gmp regulates myosin interaction with the cytoskeleton during chemotaxis of Dictyostelium
Authors: Liu Gang , Newell Peter C.
Published: April 2021

Amoebae of Dictyostelium discoideum respond to a chemotactic cyclic AMP stimulus within 10 s by the formation of an intracellular peak of cyclic GMP. In wild-type cells the cyclic GMP is rapidly degraded by a cyclic GMP-specific phosphodiesterase. In ‘streamer F’ mutants this enzyme is defective, due to mutation in the enzyme structural gene, and the cyclic GMP persists several times longer than the normal period, an effect that is correlated with a persistence in cell elongation during the chemotactic movement phase.

In this study we have used the streamer mutants NP368 and NP377, and their parental strain XP55, to study changes in cytoskeletal proteins during the chemotactic response. We have studied three proteins that change their association with the cytoskeleton after stimulation of amoebae with the chemoattractant cyclic AMP: (1) actin, (2) a protein with an apparent Mr of 190 ×103 and (3) myosin heavy chain. Both actin and the 190×103Mr protein were found to accumulate rapidly in the cytoskeleton after cyclic AMP stimulation, with a sharp peak at 5 s, and showed similar changes in the parental and streamer mutants. However, the cytoskeletal level of myosin heavy chain showed a different pattern of changes, which also clearly differed in the streamer mutants compared with the parental strain XP55. In XP55 myosin heavy chain showed an initial drop after cyclic AMP stimulation, with a trough at 3-10 s followed by a rapid rise to a sharp peak at 20–25 s. In contrast, the myosin heavy chain in the streamer mutants produced a broad peak that persisted several times longer than the parental strain.

We conclude that in the streamer mutants the defect in cyclic GMP phosphodiesterase that produces the broad peak of cyclic GMP is causally correlated with the broad peak of cytoskeletal myosin, and we suggest that this is connected with the observed phenotype of prolonged cell elongation during chemotaxis in these mutants.
Dynamics of membrane skeleton in fused red cell membranes
Authors: Cheng-Zhi Jin
Published: April 2021

The dynamic nature of the red cell membrane cytoskeleton was examined by the method of fluorescence redistribution after fusion (FRAF). Ghosts, labelled at the interior membrane surface, and intact cells, labelled at the outer surface with a different fluorophore, were fused and the passage of the fluorescent label into the unlabelled part of the membrane was followed in the fluorescence microscope. To achieve specific labelling of a cytoskeletal component only, a fluorescent analogue of phalloidin was used to label the actin. It was shown that there was no dissociation of the phalloidin during the time of the experiment, and no exchange of phalloidin between labelled and unlabelled actin in unsealed mixtures of ghosts. In the fused membrane pairs at 37 °C the phalloidin-linked fluorescence diffused in the plane of the membrane and became distributed through the membrane envelope in 1–2 h. It was shown by gel electrophoretic analysis that no detectable loss of or damage to membrane proteins resulted from the fusion process. It is concluded that, at least in membrane pairs treated with fusogen, exchange of actin or actin-containing elements occurs within the cytoskeletal network.
Establishment of plastid-based quadripolarity in spore mother cells of the moss Funaria hygrometrica
Authors: Busby C. H. , Gunning B. E. S.
Published: April 2021

Development of a tetrad of meiospores is one of the most widespread examples of geometrically precise cell morphogenesis in plants. We have studied the process in the moss Funaria hygrometrica. Changes leading to a quadripolar organization of the prophase spore mother cell (SMC) start in the archesporial cells several cell generations before meiosis. The number of plastids per cell is reduced to two and these play an increasing part in subsequent mitoses and meiosis. During meiotic prophase, the plastids elongate until they enclose the peripheral nucleus. The nucleus is then drawn back into the centre of the cell as the plastids rotate and ultimately assume a mutually perpendicular configuration. The tips of the plastids thus lie at the vertices of a tetrahedron arranged around the nucleus, which itself becomes deformed into a tetrahedral shape. Quadripolarity has now been set up in anticipation of the two meiotic divisions.

The first division spindle is also somewhat tetrahedral, with broad poles oriented perpendicular to one another along two opposite edges of the tetrahedron. As a consequence, the daughter nuclei are, from their inception, mutually perpendicular and elongated along the first spindle poles, ready for the second division, which places one haploid nucleus opposite each of the four plastid tips. Simultaneous cytokinesis then bisects the plastids and generates a tetrad of spores.

The morphological evidence thus indicates that the plastids are involved in the development of internal quadripolarity in the outwardly apolar SMCs.