bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the development of ameloblasts: I. fine structure
Authors: Katchburian E. , Holt S. J.
Published: April 2021

The cellular and fine-structural changes which take place during differentiation of ameloblasts into highly secretory cells involve growth and elongation of the cytoplasm, a change in polarity, and sequential development of organelles. The undifferentiated cells of the ameloblastic layer seem to be mainly concerned with the production of material for internal use, as they are rich in free ribosomes and have only few profiles of rough-surfaced endoplasmic reticulum. First signs of differentiation are detected when the cells begin to elongate and large numbers of microtubules oriented parallel to the cell long axis appear in the distal cytoplasm. This suggests that the microtubules provide directional guidance for the elongation of the cells. The next stage of differentiation is characterized by proliferation of the rough-surfaced endoplasmic reticulum in the distal cytoplasm and the migration of the small Golgi complex, initially at the base of the cell, to the distal cytoplasm. Intracisternal accumulation of enamel matrix-like material in the rough-surfaced endoplasmic reticulum at this stage suggests that the Golgi complex is unable to process for secretion the materials synthesized in the rough-surfaced endoplasmic reticulum. However, the presence of enamel matrix-like material in the inter-cellular spaces may indicate an attempt to secrete matrix by a mechanism which by-passes the Golgi complex. With further differentiation the Golgi complex reaches the distal cytoplasm where it develops and becomes very extensive. The appearance of enamel matrix-like material in the cisternae of the mature Golgi complex and in large secretion granules in secretory ameloblasts suggests that secretory material is processed in a manner similar to that of other exocrine cells. As soon as enamel matrix begins to be deposited at the distal surface, ameloblasts withdraw, leaving the cytoplasmic extension (Tomes process) in the early calcifying enamel. The presence of microtubules and filaments in both the early and late developing Tomes process suggests that they play a role in the directional and localized growth of this constricted cell extension. Based on previous evidence that the Tomes process has an extensive lysosomal system and on the observation that its surface membrane possesses numerous invaginations and coated pits, it is concluded that the present findings support the concept that the Tomes process – via its lysosomal vacuolar apparatus – plays a role in the changes which occur in the enamel matrix in early amelogenesis.
Roles of cortical and subcortical components in cleavage furrow formation in amphibia
Authors: Sawai Tsuyoshi
Published: April 2021

In the eggs of the newt, Triturus pyrrhogaster, 2 separate factors are recognized which take part in cleavage furrow formation. (1) The inductive capacity for the furrow formation by the cytoplasm lying under the cortex along the cleavage furrow (FIC); and (2) the reactivity of the overlying cortex to form a furrow in response to FIC.

(1) FIC. The inductive capacity is shown by the fact that FIC induces a furrow on which-ever part of the surface under which FIC is transplanted. FIC is distributed along the cleavage furrow and even extends along the future furrow plane ahead of the furrow tip. The distance FIC precedes the furrow tip is about 10 mm in the animal hemisphere and is less in the vegetal hemisphere. In the direction at right angles to the furrow plane, FIC does not spread more than 0·1 mm.

FIC is also present in the eggs of Xenopus laevis. Species specificity of FIC for induction is not found between Triturus and Xenopus.

(2) Surface layer. At the onset of the first cleavage, the reactivity of the cortex to form the furrow in answer to FIC induction is localized on the animal pole region. The reactivity of the cortex propagates medially as a belt along the surface towards the vegetal pole with the advancing tip of the cleavage furrow. After the furrow is completed, the reactivity begins to be lost from the animal pole region, and eventually over the entire surface. The reactivity, however, reappears on the animal pole region simultaneously with the second cleavage.
Cell cycle kinetics and development of Hydra attenuata: I. epithelial cells
Authors: David C. N. , Campbell R. D.
Published: April 2021

The cell cycle parameters of epithelial cells of Hydra attenuata are described. Specifically the rate of proliferation and the fraction of proliferating cells have been determined under conditions of defined growth rate. Techniques involved standard methods of cell cycle analysis using histological and tissue maceration preparations; pulse-chase and continuous labelling with [3H]thymidine followed by autoradiographic analysis, and microspectrophotometric determination of nuclear DNA content in single cells. The results indicate that more than 90% of hydra epithelial cells are actively proliferating with a cell cycle duration about equal to the tissue doubling time. In well fed hydra the average cell cycle is about 3 days long. S period is 12–15 h, G1 0–1 h, and mitosis 1·5 h. Most of the cell cycle consists of a long G2 period of variable duration (24–72 h). The results provide no evidence for a subpopulation of rapidly proliferating cells as predicted by ‘growth zone’ models of hydra morphogenesis. The results also indicate that the population of epithelial cells is self-sustaining requiring no input by differentiation from other cell types. The long and variable G2 period means that DNA synthesis and the following cell division are effectively uncoupled such that inhibitors of DNA synthesis may not stop epithelial cell division. The variable nature of the G2 period suggests it as a possible point of control of hydra growth.
Turnover of plasma membrane during phagocytosis
Authors: Goodall R. J. , Lai Y. F. , Thompson J. E.
Published: April 2021

Levels of radioactive glycine and glycerol incorporated into the plasma membrane of Acanthamoeba castellanii during phagocytosis were determined in order to elucidate how surface membrane expended during this process is replaced. The amoebae were allowed to ingest latex beads in the presence of the labelled membrane precursors and plasma membrane was then isolated and analysed for the presence of radioactivity. The isolated membrane fragments were found to be quite highly labelled. In order to ascertain whether this represented preferential synthesis of plasma membrane in response to phagocytosis, the specific radio-activities of the isolated membrane fractions were compared with those of corresponding particulate homogenates, which were composites of all cellular membranes. Enrichment values calculated in this manner proved to be essentially similar for both phagocytosing and non-phagocytosing amoebae. This indicates that de novo synthesis of plasma membrane is not essential for phagocytosis and in turn suggests that pre-existing cytoplasmic membrane is used to replace surface membrane consumed during ingestion. Presumably the incorporation of membrane precursors that was observed represents molecular turnover that occurs irrespective of phagocytosis.
Sodium-Induced Changes in the Nuclei of Monolayer HeLa Cultures
Authors: Jobst K. , Kellermayer N.
Published: April 2021

In HeLa cells cultured in a medium containing 142 mequiv./l. sodium ions only the mitotic forms are biréfringent (anisotropic index, 27/1000). In a culture medium containing 155 – 160 mequiv./l. sodium ions, large numbers of nuclear forms reminiscent of the prophase were found. In such a medium the anisotropic index was 86/1000. At low sodium ion concentration (90 – 110 mequiv./l.) these anomalous, prophase-like forms were not seen and the anisotropic index was 18/1000. The appearance of the biréfringent, prophase-like forms is related to a structural rearrangement and condensation of DNP in a hypertonic medium.
Studies on Nucleolar RNA Synthesis in Drosophila Melanogaster: II. The Influence of Conditions Resulting in a Bobbed Phenotype on Rate of Synthesis and Secondary Constriction Formation
Authors: Krider H. M. , Plaut W.
Published: April 2021

The influence of conditions resulting in bobbed phenotypes on nucleolar RNA synthesis and the formation of constrictions at nucleolus organizers was examined in larval tissues of Drosophila melanogaster. By means of [3H]uridine incorporation and autoradiographic analysis, a mutation at the bobbed locus was shown to limit the rate of nucleolar RNA synthesis in salivary glands of XO larvae. The formation of constrictions at the organizer sites of a 4-nucleolus-organizer stock was monitored in dividing neuroblast cells stained with acridine orange. Loss of the ribosomal cistrons had been reported by other workers when such stocks were maintained for several generations. In the first generation in our work, constrictions were visible at only 2 of the 4 nucleolus organizers. This situation persisted until the fifth generation, when constrictions appeared at all 4 of the organizer sites. An increase in the rate of nucleolar RNA synthesis in the salivary glands was temporally correlated with the appearance of the extra constrictions. We interpret these observations to mean that 2 of the organizers of the 4-nucleolus-organizer stock were caused to function through the loss of ribosomal RNA cistrons; thus the functional status of an organizer would appear to be subject to control.
Nucleocytoplasmic Interaction in the Eggs of Pteridium Aquilinum Maturing in the Presence of Thiouracil
Authors: Bell P. R.
Published: April 2021

In the eggs of Pteridium aquilinum maturing in the presence of a small quantity of thiouracil (0·6 × 10-3 M in the medium), known to reduce the rate of maturation by a half or more, the nuclear evaginations are particularly conspicuous. On the grounds of their structural features, their regularly narrow connexion with the nucleus, and the absence of nuclear pores from their envelopes, the evaginations are regarded as specialized formations, and not merely extensions of an amoeboid nucleus. An aggregate of electron-opaque material, probably of nucleolar origin, and believed on the basis of enzyme extractions to be largely protein, but with some ribonucleic acid, is a characteristic feature of the evaginations.

The fate of the evaginations, which disappear shortly after fertilization, remains obscure. Refined methods of fixation and staining have, however, revealed striking resemblances between the evaginations, bodies which may be detached evaginations, and mitochondria. The hypothesis that the evaginations become detached, and that some at least differentiate into mitochondria, the simplest interpretation of the events observed, has not been invalidated by this investigation.
The Long-Term Organ Culture of Tissues from Adult Amphiuma, The Congo Eel
Authors: Monnickendam Marjorie A. , Balls M.
Published: April 2021

Fragments of pancreas, liver, spleen, kidney and lung from Amphiuma means, the Congo eel, were maintained in organ culture for up to 35 days in a modified Leibovitz L15 medium. These Amphiuma tissues retained their typical histological features throughout the culture period, surviving better and for longer than tissues from other amphibian species. Liver and spleen survived better in hypotonic or isotonic media (125–230 mosmol/kg) than in hypertonic media (255–305 mosmol/kg). The mitotic incidences of these tissues in vivo were very low, and while there were no significant increases in liver, spleen and lung fragments in vitro, there were large increases in kidney and pancreas fragments. Amylase activity was found in pancreas fragments and in medium from pancreas cultures after 28 days in vitro. Amphiuma organ cultures may be useful in the in vitro study of the control of cell proliferation and cell function while normal cell interactions are maintained.
Biogenesis of Plasma Membranes in Salt Glands of Salt-Stressed Domestic Ducklings: Localization of Acyltransferase Activity
Authors: Levine A. M. , Higgins Joan A. , Barrnett R. J.
Published: April 2021

In response to salt water stress there is a marked increase in the plasma membranes of the epithelial secretory cells of the salt glands of domestic ducklings. In the present study, the fine-structural localization of the acyltransferases involved in synthesis of phospholipids has been investigated in this tissue during this increased biogenesis of plasma membranes. The specific activity of the acyltransferases of the salt gland rose in response to salt stress, and this preceded the rapid increase in weight and cellular differentiation. After the weight increase of the gland became established, the specific activity of the acyltransferases declined, but the total activity remained constant. Salt gland tissue fixed in a mixture of glutaraldehyde and formaldehyde retained 35 % of the acyltransferase activity of unfixed tissue. Cytochemical studies of the localization of acyltransferase activity in fixed and unfixed salt gland showed reaction product associated only with the lamellar membranes of the Golgi complex. This localization occurred in partially differentiated cells from salt-stressed glands to the greatest extent; and to only a small extent in cells of control tissue from unstressed salt glands. Omission of substrates resulted in absence of reaction product in association with the Golgi membranes. In addition, vesicles having limiting membranes morphologically similar to the plasma membrane occurred between the Golgi region and the plasma membrane in the partially differentiated cells. The phospholipid component of the plasma membrane appears therefore to be synthesized in association with the Golgi membranes and the membrane packaged at this site from which it moves in the form of vesicles to fuse with the pre-existing plasma membrane.
Helical Structures in the Cytoplasm of the Male Germ Cells of the Desert Locust
Authors: Gawadi Nadya
Published: April 2021

A helical structure which had previously been reported in the cytoplasm of many cell types in the desert locust, Schistocerca gregaria, was found in large quantities in the developing male germ cells. The helices were isolated from these cells and negatively stained. The helices, which lie free in the cytoplasm of spermatogonia, primary spermatocytes and secondary spermatocytes (sometimes associated with a second type of helix, a double helix), closely invest the outer surface of the Nebenkern membrane in spermatids. As the Nebenkem elongates, the helices gradually disappear and are replaced by microtubules. The mitochondrial derivatives of the Nebenkem, which form part of the sperm tail, are in late spermatids completely surrounded by a sheath of parallel microtubules.
The role of laminin-5 and its receptors in mammary epithelial cell branching morphogenesis
Authors: Stahl Stephanie , Weitzman Sigmund , Jones Jonathan C. R.
Published: April 2021

In vivo, normal mammary epithelial cells utilize hemidesmosome attachment devices to adhere to stroma. However, analyses of a potential role for hemidesmosomes and their components in mammary epithelial tissue morphogenesis have never been attempted. MCF-10A cells are a spontaneously immortalized line derived from mammary epithelium and possess a number of characteristics of normal mammary epithelial cells including expression of hemidesmosomal associated proteins such as the two bullous pemphigoid antigens, α6β4 integrin and its ligand laminin-5. More importantly, MCF-10A cells readily assemble mature hemidesmosomes when plated onto uncoated substrates. When maintained on matrigel, like their normal breast epithelial cell counterparts, MCF-10A cells undergo a branching morphogenesis and assemble hemidesmosomes at sites of cell-matrigel interaction. Function blocking antibodies specific for human laminin-5 and the α subunits of its two known receptors (α3β1 and α6β4 integrin) not only inhibit hemidesmosome assembly by MCF-10A cells but also impede branching morphogenesis induced by matrigel. Our results imply that the hemidesmosome, in particular those subunits comprising its laminin-5/integrin ‘backbone’, play an important role in morphogenetic events. We discuss these results in light of recent evidence that hemidesmosomes are sites involved in signal transduction.
Studies on the eel sperm flagellum: I. The structure of the inner dynein arm complex
Authors: Woolley D. M.
Published: April 2021

The highly motile, 9 + 0 sperm axoneme of Anguilla has inner dynein arms (IDAs) but not outer dynein arms. The in situ morphology of these IDAs is shown here to be essentially identical to the IDA morphology already seen in the axonemes of Chlamydomonas, Tetrahymena and Beroë, and in the sperm tails of echinoderms and several vertebrate species. In addition, this study demonstrates: (1) that the nexin (circumferential) links are present in Anguilla and are typical; (2) that IDA1 incorporates an archway, supported by a pillarstructure; (3) that images from thin sections and whole mounts are consistent with those from replicas of rapidly-frozen specimens; and (4) that the IDA and nexin link morphology is apparently unaffected by whether the axoneme is depleted of ATP, relaxed with ATP and vanadate, or inhibited by high ATP. An attempt has been made to reconcile the emergent morphology of the IDA complex with all earlier descriptions in the literature. From a detailed comparison of the results with published information on Chlamydomanas mutants, it is concluded that the nexin (circumferential) link is a major part of the ‘dynein regulatory complex’.
Histidine kinases in signal transduction pathways of eukaryotes
Authors: Loomis William F. , Shaulsky Gad , Wang Nancy
Published: April 2021

Autophosphorylating histidine kinases are an ancient conserved family of enzymes that are found in eubacteria, archaebacteria and eukaryotes. They are activated by a wide range of extracellular signals and transfer phosphate moieties to aspartates found in response regulators. Recent studies have shown that such two-component signal transduction pathways mediate osmoregulation in Saccharomyces cerevisiae, Dictyostelium discoideum and Neurospora crassa. Moreover, they play pivotal roles in responses of Arabidopsis thaliana to ethylene and cytokinin. A transmembrane histidine kinase encoded by dhkA accumulates when Dictyostelium cells aggregate during development. Activation of DhkA results in the inhibition of its response regulator, RegA, which is a cAMP phosphodiesterase that regulates the cAMP dependent protein kinase PKA. When PKA is activated late in the differentiation of prespore cells, they encapsulate into spores. There is evidence that this two-component system participates in a feedback loop linked to PKA in prestalk cells such that the signal to initiate encapsulation is rapidly amplified. Such signal transduction pathways can be expected to be found in a variety of eukaryotic differentiations since they are rapidly reversible and can integrate disparate signals.
Changes in histone synthesis and modification at the beginning of mouse development correlate with the establishment of chromatin mediated repression of transcription
Authors: Wiekowski Maria , Miranda Miriam , Nothias Jean-Yves , DePamphilis Melvin L.
Published: April 2021

The transition from a late 1-cell mouse embryo to a 4-cell embryo, the period when zygotic gene expression begins, is accompanied by an increasing ability to repress the activities of promoters and replication origins. Since this repression can be relieved by either butyrate or enhancers, it appears to be mediated through chromatin structure. Here we identify changes in the synthesis and modification of chromatin bound histones that are consistent with this hypothesis. Oocytes, which can repress promoter activity, synthesized a full complement of histones, and histone synthesis up to the early 2-cell stage originated from mRNA inherited from the oocyte. However, while histones H3 and H4 continued to be synthesized in early 1-cell embryos, synthesis of histones H2A, H2B and H1 (proteins required for chromatin condensation) was delayed until the late 1-cell stage, reaching their maximum rate in early 2-cell embryos. Moreover, histone H4 in both 1-cell and 2-cell embryos was pre-dominantly diacetylated (a modification that facilitates transcription). Deacetylation towards the unacetylated and monoacetylated H4 population in fibroblasts began at the late 2-cell to 4-cell stage. Arresting development at the beginning of S-phase in 1-cell embryos prevented both the appearance of chromatin-mediated repression of transcription in paternal pronuclei and synthesis of new histones. These changes correlated with the establishment of chromatin-mediated repression during formation of a 2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates and core histones are deacetylated.
Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells
Authors: Parton R. M. , Fischer S. , Malhó R. , Papasouliotis O. , Jelitto T. C. , Leonard T. , Read N. D.
Published: April 2021

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cyto-plasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cyto-plasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pHsensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between ±0.03 and ±0.06 of a pH unit and a spatial resolution of 2.3 μm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (ΔpH of >0.1 unit) was observed within the apical 50 μm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.
Tip localized Ca2+ Pulses are coincident with peak pulsatile growth rates in pollen tubes of Lilium longiflorum
Authors: Messerli Mark , Robinson Kenneth R.
Published: April 2021

It is known that locally elevated Ca2+ at the growing tips of pollen tubes is necessary for pollen tube elongation. Here we show that this localized Ca2+ is also temporally regulated and is closely associated with pulsatile tip growth. Lilium longiflorum pollen tubes were injected with the photoprotein, aequorin, and the Ca2+-dependent light output was detected with a low noise photon-counting system. Ca2+ pulses with a mean period of 40 seconds were invariably associated with growth. The pulses were sporadic and of low amplitude for about the first 1.5 hours after germination. With subsequent growth, pulses increased in amplitude and the period between pulses became more regular. We have localized these Ca2+ pulses to the elongating end of the growing tube. The Ca2+ pulses are asymmetrical, rising more slowly than they fall. We estimate that the Ca2+ concentration at the peak of the pulses reaches nearly 10 μM. The addition of 100 μM La3+, a Ca2+ channel blocker, extinguished the pulses. An analysis of growth of elongating tubes establishes that extension is pulsatile, with a 42 second period between pulses. Calcium imaging, using the fluorescent indicator, Calcium Green dextran, shows that calcium pulses are coincident with peak growth rates.
Changing intestinal connective tissue interactions alters homeobox gene expression in epithelial cells
Authors: Lorentz Isabelle Duluc, Olivier , Fritsch Christine , Michèle Kedinger Cathy Leberquier , Freund Jean-Noël
Published: April 2021

In segmented organs, homeobox genes are involved in axial patterning and cell identity. Much less is known about their role in non-segmented endoderm derivatives such as the digestive epithelium. Using a xenograft model of fetal intestinal anlagen implanted under the skin of nude mice, we have investigated whether the expression of five homeobox genes (HoxA-4, HoxA-9, HoxC-8, Cdx-1 and Cdx-2) is modified when intestinal epithelium undergoes normal development or displays heterodifferentiation in association with heterotopic mesenchyme. In homotypic associations of fetal endoderm and mesenchyme that recapitulate normal development, the overall pattern of homeobox gene expression was maintained: HoxA-9 and HoxC-8 were the highest in the colon and ileum, respectively, and HoxA-4 was expressed all along the intestine; Cdx-1 and Cdx-2 exhibited an increasing gradient of expression from small intestine to colon. Yet, grafting per se caused a faint upregulation of HoxA-9 and HoxC-8 in small intestinal regions in which these genes are not normally expressed, while the endoderm-mesenchyme dissociation-association step provoked a decay of Cdx-1 in the colon. In heterotopic associations of colonic endoderm with small intestinal mesenchyme, the colonic epithelium exhibited heterodifferentiation to a small intestinal-like phenotype. In this case, we observed a decay of HoxA-9 expression and an upregulation of HoxC-8. Additionally, heterodifferentiation of the colonic epithelium was accompanied by a downregulation of Cdx-1 and Cdx-2 to a level similar to that found in the normal small intestine. To demonstrate that mesenchyme-derived cells can influence Cdx-1 and Cdx-2 expression in the bowel epithelium, fetal jejunal endoderm was associated with intestinal fibroblastic cell lines that either support small intestinal-like or colonic-like morphogenesis. A lower expression of both homeobox genes was shown in grafts presenting the small intestinal phenotype than in those showing glandular colonic-like differentiation. Taken together, these results suggest that homeobox genes participate in the control of the positional information and/or cell differentiation in the intestinal epithelium. They also indicate that the level of Cdx-1 and Cdx-2 homeobox gene expression is influenced by epithelial-mesenchymal cell interactions in the intestinal mucosa.
Functional expression of the alpha 7 integrin receptor in differentiated smooth muscle cells
Authors: Yao Chung-Chen , Breuss Johannes , Pytela Robert , Kramer Randall H.
Published: April 2021

Expression of the α7 integrin is developmentally regulated and is thought to be tissue-specific for both skeletal and cardiac muscles. We now report that α7 is also strongly and ubiquitously expressed by various types of smooth muscle, including vascular, gastrointestinal and genitourinary smooth muscles. In addition, α7 was surface-expressed by a number of smooth muscle cell lines that maintained their differentiated phenotype following adaptation to culture. Studies with the mouse 9E11G smooth muscle cell line showed that the α7 integrin mediated both adhesion and motility of these cells on laminin 1 substrates. α7 expression appears to correlate with the smooth-muscle-differentiated phenotype. The multipotential P19 mouse embryonic stem cell line lacks α7 but uses the α6 integrin to adhere to laminin 1. Following retinoic acid-induced P19 differentiation predominantly to the smooth muscle cell lineage, high expression of α7 was detected along with partial dependence on α7 for binding to laminin. The expression of α7 paralleled the induction of smooth-muscle-specific α-actin, as revealed by dual-labeling flow cytometry. In contrast, α7, which initially was highly expressed on the surface of vascular smooth muscle cell explants, was rapidly downregulated in smooth muscle cell outgrowths as they dedifferentiated into their synthetic phenotype. The results indicate that the expression of α7 integrin in smooth muscle cells is associated with their differentiated phenotype and mediates their interaction with laminins.
Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent ‘envelope smoothing’ event
Authors: Wiese Christiane , Goldberg Martin W. , Allen Terence D. , Wilson Katherine L.
Published: April 2021

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying (‘rough’) vesicles, many of which bound directly to chromatin, and ‘smooth’ vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatinattachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed ‘envelope smoothing’ occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that ‘smoothing’ required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.
The anaphase-promoting complex is required in G1 arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S-phase
Authors: Irniger Stefan , Nasmyth Kim
Published: April 2021

Inactivation of B-type cyclin dependent kinases due to ubiquitin-mediated cyclin proteolysis is necessary for the exit from mitosis. In Saccharomyces cerevisiae, proteolysis is initiated at the onset of anaphase and remains active until Cln1 and Cln2 cyclins appear in late G1 of the subsequent cell cycle. A large particle called the anaphase-promoting complex (APC) which is composed of the TPR proteins Cdc16p/Cdc23p/Cdc27p and other proteins is required for B-type cyclin ubiquitination in both anaphase and during G1 phase. The APC has an essential role for the separation of sister chromatids and for the exit from mitosis, but until now it was unclear whether the persistence of APC activity throughout G1 had any physiological role. We show here that the APC is needed in G1 arrested cells to inhibit premature appearance of B-type cyclins and to prevent unscheduled initiation of DNA replication. When pheromone arrested cells of cdc16 and cdc23 mutants were shifted to the restrictive temperature, they underwent DNA replication in the presence of pheromone. DNA replication also occurred in a G1 arrest induced by G1 cyclin (Cln) depletion, indicating that mutant cells with a defective APC initiate DNA replication without the Cln G1 cyclins, which are normally needed for the onset of S-phase. Degradation of Clb2p, Clb3p and Clb5p depends on the APC. This suggests that accumulation of any one of the six B-type cyclin proteins could account for the precocious replication of cdc16 and cdc23 mutants.
In vitro exocytosis in sea urchin eggs requires a synaptobrevin-related protein
Authors: Avery Julia , Hodel Alois , Whitaker Michael
Published: April 2021

Sea urchin eggs provide an efficient in vitro model of exocytosis. We have identified proteins in sea urchin eggs that cross-react with antibodies to mammalian synaptobrevin, synaptotagmin, SNAP-25, syntaxin and rab3a. We show that these proteins are localized to the sea urchin egg cortex, using western blotting and immunocytochemistry. Tetanus toxin light chain cleaves the synaptobrevin-related protein in vitro and inhibits calcium-induced exocytosis. These data demonstrate a conservation between phyla of protein sequence and molecular mechanisms thought to facilitate exocytosis and show that the sea urchin egg provides a unique in vitro exocytotic model with which to study the conserved protein machinery of membrane fusion during secretion.
Occludin as a possible determinant of tight junction permeability in endothelial cells
Authors: Hirase Tetsuaki , Staddon James M. , Saitou Mitinori , Ando-Akatsuka Yuhko , Itoh Masahiko , Furuse Mikio , Fujimoto Kazushi , Tsukita Shoichiro , Rubin Lee L.
Published: April 2021

Endothelial cells provide a crucial interface between blood and tissue environments. Free diffusion of substances across endothelia is prevented by the endothelial tight junction, the permeability of which varies enormously depending on tissue. Endothelial cells of the blood-brain barrier possess tight junctions of severely limited permeability, whereas those of non-neural tissue are considerably leakier, but the molecular basis for this difference is not clear. Occludin is a major transmembrane protein localizing at the tight junction. In this study, we show, by immunocytochemistry, that occludin is present at high levels and is distributed continuously at cell-cell contacts in brain endothelial cells. In contrast, endothelial cells of non-neural tissue have a much lower expression of occludin, which is distributed in a discontinuous fashion at cell-cell contacts. The apparent differences in occludin expression levels were directly confirmed by immunoblotting. The differences in occludin protein were reflected at the message level, suggesting transcriptional regulation of expression. We also show that occludin expression is developmentally regulated, being low in rat brain endothelial cells at postnatal day 8 but clearly detectable at post-natal day 70. Our data indicate that regulation of occludin expression may be a crucial determinant of the tight junction permeability properties of endothelial cells in different tissues.
The neural crest population responding to endothelin-3 in vitro includes multipotent cells
Authors: Stone Joanne G. , Spirling Lucy I. , Richardson Michael K.
Published: April 2021

The peptide endothelin 3 (EDN3) is essential for normal neural crest development in vivo, and is a potent mitogen for quail truncal crest cells in vitro. It is not known which subpopulations of crest cells are targets for this response, although it has been suggested that EDN3 is selective for melanoblasts. In the absence of cell markers for different precursor types in the quail crest, we have characterised EDN3-responsive cell types using in vitro colony assay and clonal analysis. Colonies were analysed for the presence of Schwann cells, melanocytes, adrenergic cells or sensory-like cells. We provide for the first time a description of the temporal pattern of lineage segregation in neural crest cultures. In the absence of exogenous EDN3, crest cells pro-liferate and then differentiate. Colony assay indicates that in these differentiated cultures few undifferentiated pre-cursors remain and there is a low replating efficiency. By contrast, in the presence of 100 ng/ml EDN3 differentiation is inhibited and most of the cells maintain the ability to give rise to mixed colonies and clones containing neural crest derivatives. A high replating efficiency is maintained. In secondary culture there was a progressive decline in the number of cell types per colony in control medium. This loss of developmental potential was not seen when exogenous EDN3 was present. Cell type analysis suggests two novel cellular targets for EDN3 under these conditions. Contrary to expectations, one is a multipotent precursor whose descendants include melanocytes, adrenergic cells and sensory-like cells; the other can give rise to melanocytes and Schwann cells. Our data do not support previous claims that the action of EDN3 in neural crest culture is selective for cells in the melanocyte lineage.
Cell-type specific calcium signalling in a Drosophila epithelium
Authors: Rosay Philippe , Davies Shireen A. , Yu Yong , Sözen M. Ali , Kaiser Kim , Dow Julian A. T.
Published: April 2021

Calcium is a ubiquitous second messenger that plays a critical role in both excitable and non-excitable cells. Calcium mobilisation in identified cell types within an intact renal epithelium, the Drosophila melanogaster Malpighian tubule, was studied by GAL4-directed expression of an aequorin transgene. CAP2b, a cardioactive neuropeptide that stimulates fluid secretion by a mechanism involving nitric oxide, causes a rapid, dosedependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. Furthermore, the data suggest a role for calcium signalling in the modulation of the nitric oxide signalling pathway in this epithelium. The GAL4-targeting system allows general application to studies of cell-signalling and pharmacology that does not rely on invasive or cytotoxic techniques.
Normal development of preimplantation mouse embryos deficient in gap junctional coupling
Authors: Sousa Paul A. De , Juneja Subhash C. , Caveney Stanley , Houghton Franchesca D. , Davies Tyler C. , Reaume Andrew G. , Rossant Janet , Kidder Gerald M.
Published: April 2021

The connexin multigene family (13 characterized members in rodents) encodes the subunits of gap junction channels. Gap junctional intercellular coupling, established during compaction of the preimplantation mouse embryo, is assumed to be necessary for development of the blastocyst. One member of the connexin family, connexin43, has been shown to contribute to the gap junctions that form during compaction, yet embryos homozygous for a connexin43 null mutation develop normally, at least until implantation. We show that this can be explained by contributions from one or more additional connexin genes that are normally expressed along with connexin43 in preimplantation development. Immunogold electron microscopy confirmed that roughly 30% of gap junctions in compacted morulae contain little or no connexin43 and therefore are likely to be composed of another connexin(s). Confocal immunofluorescence microscopy was then used to demonstrate that connexin45 is also assembled into membrane plaques, beginning at the time of compaction. Correspondingly, embryos homozygous for the connexin43 null mutation were found to retain the capacity for cell-to-cell transfer of fluorescent dye (dye coupling), but at a severely reduced level and with altered permeability characteristics. Whereas mutant morulae showed no evidence of dye coupling when tested with 6-carboxyfluorescein, dye coupling could be demonstrated using 2′,7′-dichlorofluorescein, revealing permeability characteristics previously established for connexin45 channels. We conclude that preimplantation development in the mouse can proceed normally even though both the extent and nature of gap junctional coupling have been perturbed. Despite the distinctive properties of connexin43 channels, their role in preimplantation development can be fulfilled by one or more other types of gap junction channels.
Endocytic pathway from the basal plasma membrane to the ruffled border membrane in bone-resorbing osteoclasts
Authors: Palokangas Harri , Mulari Mika , Väänänen H. Kalervo
Published: April 2021

We have characterized the convoluted ruffled border (RB) membrane that an activated osteoclast maintains against the bone matrix. The bulk of both lgp110 and rab7, a small GTP-binding protein participating in vesicle fusion to late endosomes, was localized to the RB. This indicates that the membrane has some characteristics of late endosomal membranes in other cells. Furthermore, the bulk of membrane-bound rab7 on the RB suggests that endocytic membrane transport is oriented towards the RB in resorbing osteoclasts. Consistently, both lumenal horseradish peroxidase and receptor-bound transferrin, a marker of the early endosomal recycling pathway, were efficiently endocytosed from the basal plasma membrane and delivered to the RB. Delivery of membrane-associated transferrin to the RB further indicates that the RB is compositionally different from lysosomes and suggests that the endocytic pathway contributes to the maintenance of functional RB. In addition to transporting receptor-bound cargo to the RB, the endocytic pathway could act in balancing the membrane traffic associated with transcytosis from the RB to the basal plasma membrane. Endocytic processes (retrieval of mannose 6-phosphate receptors) in osteoclasts appeared to be fairly sensitive to bafilomycin A1, a specific inhibitor of vacuolar-type proton ATPases. Thus blocking the endocytic membrane traffic towards the RB could explain the inactivation of cells by low concentrations of the drug.
Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation
Authors: Moser Michael J. , Flory Mark R. , Davis Trisha N.
Published: April 2021

The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP). In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome. Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Cam1p) in fission yeast, where calmodulin had not been localized by any method. We find GFP-Cam1p also localizes both to sites of polarized cell growth and to the fission yeast SPB. The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant. Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis. Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed. Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells. Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.
DNA damage inhibits proteolysis of the B-type cyclin Clb5 in S. cerevisiae
Authors: Germain Doris , Hendley Joy , Futcher Bruce
Published: April 2021

Cell cycle progression is mediated by waves of specific cyclin dependent kinases (CDKs) in all eukaryotes. Cyclins are degraded by the ubiquitin pathway of proteolysis. The recent identification of several components of the cyclin proteolysis machinery has highlighted both the importance of proteolysis at multiple transition points in the cell cycle and the involvement of other substrates degraded by the same machinery. In this study, we have investigated the effects of DNA damage on the cyclin proteolytic machinery in Saccharomyces cerevisiae. We find that the half-life of the B-type cyclin Clb5 is markedly increased following DNA damage while that of G1 cyclins is not. This effect is independent of cell cycle phase. Clb5 turnover requires p34CDC28 activity. Stabilisation of Clb5 correlates with an increase in tyrosine phosphorylation of p34CDC28, but stabilisation does not require this tyrosine phosphorylation. The stabilisation is independent of the checkpoint genes Mec1 and Rad53. These observations establish a new link between the regulation of proteolysis and DNA damage.
Aggregation reroutes molecules from a recycling to a vesicle-mediated secretion pathway during reticulocyte maturation
Authors: Vidal Michel , Mangeat Paul , Hoekstra Dick
Published: April 2021

Endocytosis of the Tf/TfR complex is essentially the only pathway active in maturing reticulocytes, while exosomes, formed by invagination of the endosomal membrane, provide a mechanism to eliminate seemingly obsolescent proteins, including the TfR, when their function is completed. In this study, we examined molecular trafficking in the recycling and exosome-directed pathways during endocytosis in maturing reticulocytes. To this end, the flow of two exogenously inserted fluorescent lipid analogs, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]) sphingomyelin (C6-NBD-SM) and N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) was monitored and compared to that of the transferrin (Tf)/Tf receptor (TfR) complex. Prior to elimination via exosomes, the TfR actively recycles with a half-time of approx. 2 minutes. The recycling kinetics of C6-NBD-SM, as bulk plasma membrane marker, are identical to those of the apoTf/TfR complex, as shown by fluorescence microscopy and biochemical analysis. By contrast, although efficiently internalized along the same pathway, N-Rh-PE does not return to the cell surface. More specifically, sucrose gradient analysis and immunoisolation experiments demonstrated that N-Rh-PE accumulates in exosomes, which are eventually released into the extracellular medium. Fluorometric measurements showed that exogenously inserted N-Rh-PE is present in the reticulocyte plasma membrane as small molecular clusters. Moreover, a close correlation was observed between the fate of crosslinked proteins, including the TfR and acetylcholinesterase (AChE), and the fate of the clustered lipid N-Rh-PE. Thus antibody-induced aggregation of specific proteins like the TfR and AChE, which are normally sorted into exosomes during reticulocyte maturation, enhances their shedding by the exosomal pathway. Taken together, the results support the hypothesis that aggregation of either proteins or lipids act as a general sorting signal for exosomal processing, thereby inhibiting reentry in a recycling pathway and providing an effective means for clearing molecules from the cell surface and their eventual elimination from the cells.