bluuebunny/crossref_metadata_2025 huggingface dataset:
The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility
Authors: Gagnon Claude , White Daniel , Cosson Jacky , Huitorel Philippe , Eddé Bernard , Desbruyères Elisabeth , Paturle-Lafanechère Laurence , Multigner Luc , Job Didier , Cibert Christian
Published: April 2021

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly α-tubulin in sea urchin sperm axonemes and equally well brain α- and β-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of α-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated α-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of α-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate.

Taken together, the data reported here indicate that the polyglutamylated lateral chain of α-tubulin plays a dynamic role in a dynein-based motility process.
Regulation of cell migration by the integrin β subunit ectodomain
Authors: Filardo Edward J. , Deming Sandra L. , Cheresh David A.
Published: April 2021

CS-1 melanoma cells transfected with cDNAs encoding either the β3 or β5 integrin subunit protein express αvβ3 or αvβ5, respectively, enabling them to adhere to vitronectin yet only αvβ3 promotes cell spreading and migration on this substrate. Following exposure to insulin or insulin-like growth factor, αvβ5-expressing CS-1 cells gain the ability to migrate on vitronectin. To identify structural regions in β3 or β5 that account for these distinct biological properties, CS-1 cells were transfected with one of two chimeric β subunit proteins, in which the ectoand cytoplasmic domains of β3 and β5 were exchanged (termed αvβ3/5 or αvβ5/3). Surprisingly, αvβ3/5 expressing cells spread and migrate on vitronectin while cells expressing αvβ5/3 do not unless they are exposed to cytokine. These findings suggest that the distinct migratory properties mediated by integrins αvβ3 and αvβ5 and their response to cytokine activation is determined by a sequence(s) within the ectodomain of the integrin β subunit.
Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells
Authors: Mayer Arie , Ivanov Ivan E. , Gravotta Diego , Adesnik Milton , Sabatini David D.
Published: April 2021

An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15-30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-γ-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane.
Deficiency of the integrin β4 subunit in junctional epidermolysis bullosa with pyloric atresia: consequences for hemidesmosome formation and adhesion properties
Authors: Niessen Carien M. , van der Raaij-Helmer Liesbeth M. H. , Hulsman Esther H. M. , van der Neut Ronald , Jonkman Marcel F. , Sonnenberg Arnoud
Published: April 2021

Junctional epidermolysis bullosa (JEB) comprises a group of inherited autosomal recessive blistering disorders characterized by dermo-epidermal separation through the lamina lucida of the basement membrane. We identified a patient with JEB associated with pyloric atresia (PA), in whom the integrin β4 subunit was completely absent. At the ultrastructural level, the hemidesmosomes were reduced in number, appeared rudimentary and lacked a subbasal dense plate and frequently an inner attachment plaque. However, keratin filaments were still anchored to the cytoplasmic plaque of the hemidesmosome. Immunofluorescence analysis showed that the β4 subunit was absent in the skin of the PA-JEB patient, whereas the α6 subunit appeared to be normally distributed along the basement membrane zone, as were the other hemidesmosomal components BP230, BP180 and HD1. Furthermore, the α3 and β1 subunits were not only detected at the lateral membranes of basal cells in PA-JEB skin, as in normal skin, but also along the basement membrane zone. The few hemidesmosome-like structures found in cultured keratinocytes from the PA-JEB patient contained the hemidesmosomal components BP230, BP180 and HD1, but not the integrin α6 subunit. Like α3, this subunit was colocalized with vinculin in focal contacts at the ends of actin stress fibers. Immunoprecipitation analysis revealed that α6 was associated with β1 on PA-JEB keratinocytes, whereas normal human keratinocytes (NHKs) exclusively express α6β4 on their cell surface. The initial adhesion of PA-JEB and normal keratinocytes to laminin-1 and laminin-5, both ligands for α6β1 and α6β4, was similar. In migration assays, the PA-JEB keratinocytes were more motile on laminin-5 than normal keratinocytes. Our observations indicate that the integrin α6β4 plays a crucial role in the proper assembly of hemidesmosomes and in the stabilization of the dermal-epidermal junction. The fragility of the skin and the blistering in this patient appear to have been due to the deficiency of the integrin β4 subunit, which results in the formation of too few and structurally abnormal hemidesmosomes.
Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system
Authors: Soussi-Yanicostas Nadia , Hardelin Jean-Pierre , Arroyo-Jimenez Maria del Mar , Ardouin Olivier , Legouis Renaud , Levilliers Jacqueline , Traincard François , Betton Jean-Michel , Cabanié Lucien , Petit Christine
Published: April 2021

The KAL gene is responsible for the X-chromosome linked form of Kallmann’s syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.
Transport of insulin and albumin by the microvascular endothelium of the rete mirabile
Authors: Bendayan Moïse , Rasio Eugenio A.
Published: April 2021

Vascular permeability for albumin and insulin in the continuous capillary network of the rete mirabile of the eel swimbladder was evaluated by ultrastructural immunocytochemistry and countercurrent perfusion experiments. Upon perfusion of the rete capillaries with a buffer solution containing albumin and insulin, these serum proteins were revealed at the electron microscope level, by the Protein A-gold immunocytochemical technique on a post-embedding step. For the simultaneous detection of both proteins, the double labeling technique with different sized gold particles was used. Furthermore, labeling was performed with the mixture of anti-albumin and anti-insulin antibodies. The labelings obtained were morphometrically evaluated and demonstrate that: (1) serum proteins such as albumin and insulin are transported by the endothelial cells through their plasmalemmal vesicular system; (2) insulin is transported preferentially to albumin; and (3) this transport involves different populations of plasmalem-mal vesicles. Measurements of diffusion permeability coefficients have confirmed the preferential transport of insulin, its coefficient being higher than that of albumin. Conversely, when compared to that of inulin or sucrose, which are assumed to be markers of the paracellular diffusion, it was found to be much lower, indicating that transcytosis through the vesicular system is less efficient than diffusion along the intercellular junctions. These results indicate that transcytosis of insulin and albumin occurs via different sets of plasmalemmal vesicles, probably through receptor-mediated mechanisms, and that the overall rate of transport across the rete capillaries, with respect to paracellular diffusion, is higher for insulin than for albumin.
The Schizosaccharomyces pombe pyp1 protein tyrosine phosphatase negatively regulates nutrient monitoring pathways
Authors: Santo Paola Dal , Blanchard Barbara , Hoffman Charles S.
Published: April 2021

The Schizosaccharomyces pombe pyp1+ gene, encoding a protein tyrosine phosphatase (pyp1), was isolated as a high copy number suppressor of a mutation that results in reduced cAMP-dependent protein kinase (PKA) activity. Overexpression of pyp1+ inhibits both transcription of the fbp1 gene, which is negatively regulated by a glucoseinduced activation of PKA, and sexual development, which is negatively regulated by PKA through a nitrogen- and glucose-monitoring mechanism. Overexpression of a catalytically inactive form of pyp1 has little effect on either process. Previous studies suggest that overexpression of pyp1+ results in a mitotic delay by positively regulating wee1 activity. We show that pyp1 repression of fbp1 transcription is independent of wee1. The direct role of the pyp1 protein is to dephosphorylate and inactivate the sty1/spc1 mitogen-activated protein kinase (MAPK) that is activated by the wis1 MAPK kinase. As overexpression of pyp1+ has no further effect upon the mitotic delay observed in a wis1 deletion strain, the role of pyp1 appears to be restricted to negative regulation of the sty1/spc1 MAPK. This study indicates that pyp1 negatively regulates fbp1 transcription, sexual development and mitosis by inactivation of the sty1/spc1 MAPK, but that bifurcations downstream of the MAPK separate these processes as seen by the differential role for the wee1 gene.
The wis1 signal transduction pathway is required for expression of cAMP-repressed genes in fission yeast
Authors: Stettler Sophie , Warbrick Emma , Prochnik Simon , Mackie Shaun , Fantes Peter
Published: April 2021

The wis1 protein kinase of Schizosaccharomyces pombe is a member of the MAP kinase kinase family. Loss of wis1 function has previously been reported to lead to a delay in the G2-mitosis transition, loss of viability in stationary phase, and hypersensitivity to osmotic shock. It acts at least in part by activating the MAP kinase homologue sty1; loss-of-function sty1 mutants share many phenotypes with wis1 deletion mutants.

We show here that, in addition, loss of wis1 function leads to defective conjugation, and to suppression of the hyperconjugation phenotype of the pat1-114 mutation. Consistent with this, the induction of the mei2 gene, which is normally induced by nitrogen starvation, is defective in wis1 mutants. In wild-type cells, nitrogen starvation leads to mei2 induction through a fall in intracellular cyclic AMP (cAMP) level and activity of the cAMP-dependent protein kinase. We show here that wis1 function is required for mei2 induction following nitrogen starvation. Expression of the fbp1 gene is negatively regulated by cAMP in response to glucose limitation: induction of fbp1 also requires wis1 and sty1 function. Loss of wis1 is epistatic over increased fbp1 expression brought about by loss of adenylate cyclase (git2/cyr1) or cAMP-dependent protein kinase (pka1) function. These observations can be explained by a model in which the pka1 pathway negatively regulates the wis1 pathway, or the two pathways might act independently on downstream targets. The latter explanation is supported, at least as regards regulation of cell division, by the observation that loss of function of the regulatory subunit of the cAMP-dependent protein kinase (cgs1) brings about a modest increase in cell length at division in both wis1+ and wis1Δ genetic backgrounds.
Calcium mobilization and influx during sperm exocytosis
Authors: Spungin Ben , Breitbart Haim
Published: April 2021

We have previously shown that two intracellular events which occur during capacitation of bovine sperm are the formation of actin filaments on the plasma and outer acrosomal membranes and the attachment of a PIP2-specific phospholipase C (PLC) to this membrane bound F-actin. This PLC plays an essential role in sperm exocytosis (acrosome reaction). In the present report, we further elucidated the role of this PLC using a PIP2-specific PLC of bacterial origin. This PLC is different from the endogenous sperm PLC in that it is calcium independent and not inhibited by neomycin. Here we report using bovine sperm that this bacterial PLC can restore actin release from extracted membranes as well as membrane fusion in a cellfree assay when the endogenous PLC is inhibited by neomycin. The sperm PLC requires 2 μM calcium for half maximal activation, while half maximal actin release from extracted plasma membranes occurs at 80 μM. Extracted sperm membranes were examined for calcium pumps and channels. Sperm plasma membranes were found to possess a thapsigargin insensitive calcium pump and calcium channels which are opened by phosphorylation by protein kinase C. The acrosomal membrane possesses a calcium pump which is inhibited by thapsigargin and calcium channels which are opened by cAMP. These observations are discussed in terms of a model of acrosomal exocytosis which involves a calcium rise that occurs in two stages resulting from calcium mobilization from internal stores followed by influx of extracellular calcium.
Migration of highly aggressive melanoma cells on hyaluronic acid is associated with functional changes, increased turnover and shedding of CD44 receptors
Authors: Goebeler Matthias , Kaufmann Dieter , Bröcker Eva-Bettina , Klein C. Eberhard
Published: April 2021

Recent evidence indicates that CD44, a multifunctional adhesion receptor involved in cell-cell as well as in cell-matrix interactions, plays an important role in local progression and metastasis of malignant tumors. We have studied a set of human melanoma cell lines differing in their metastatic potential in nude mice as well as in normal melanocytes for changes in CD44 expression and function. All melanocytes and melanoma cell lines tested highly expressed the CD44 standard form (CD44s, 85 kDa) but variants at low levels only. With respect to one of the CD44-associated functions primarily involved in tumor progression we found that two highly metastatic tumor cell lines, MV3 and BLM, showed fivefold higher migration rates towards hyaluronate than melanomas with low metastatic potential and normal melanocytes. Moreover, the highly metastatic cell lines expressed fourto sixfold higher levels of the CD44 epitope involved in hyaluronic acid-binding (monoclonal antibody Hermes-1) than less aggressive melanomas and melanocytes. Hermes-1 efficiently blocked haptotaxis to hyaluronate, supporting the functional relevance of this epitope. In contrast, expression levels of other CD44s epitopes recognized by seven different anti-CD44 monoclonal antibodies were unchanged, suggesting that the migratory behaviour of the cells depends on the formation of the hyaluronate-binding Hermes-1 epitope rather than on the overall CD44s surface expression, which was virtually identical in all melanoma and melanocyte cell lines tested. Differences in the accessibility of the hyaluronate-binding epitope defined by Hermes-1 correlated with the phosphorylation state of CD44s, probably reflecting different activation states of the receptor. Furthermore, immunoprecipitation and pulse/chase studies revealed a threeto fivefold increase in CD44 synthesis in the highly aggressive melanoma cells as compared to the other cell lines and the melanocytes, indicating a reduction of CD44 half-life and up-regulation of turnover. Moreover, highly aggressive melanoma cell lines were found to shed significant amounts of CD44 from the cell surface and to secrete its ligand hyaluronic acid, which may refer to an ‘autocrine’ mechanism mediating melanoma cell motility.
Changes in expression of monoclonal antibody epitopes on laminin-5r induced by cell contact
Authors: Plopper George , Falk-Marzillier Jutta , Glaser Scott , Fitchmun Mark , Giannelli Gianluigi , Romano Tracy , Jones Jonathan C. R. , Quaranta Vito
Published: April 2021

Laminin-5r is a basement membrane component that promotes rapid adhesion and hemidesmosome formation in epithelial cells. We raised monoclonal antibodies and identified their corresponding epitopes on the constituent chains of laminin-5r by western blotting. Using a combination of immunoprecipitation and ELISA assays, we determined that these epitopes are differentially exposed on two forms of the laminin-5r heterotrimer: soluble (passively adsorbed onto plastic) and cell-associated. Antibody 5C5 epitope is exposed on the cell-associated form, but not the soluble/passively adsorbed form of laminin-5r. Epitopes reactive with antibodies CM6, FM3, and TR1 are also preferentially exposed on cell-associated laminin-5r, such that reactivity of these antibodies with the cell-associated form is fourfold higher than with the soluble/passively adsorbed form in ELISA assays. Incubation of passively adsorbed laminin-5r with the human epithelial cell line SCC12 induced exposure of 5C5 and CM6, FM3, or TR1 epitopes. These data suggest that cells actively modify laminin-5r, perhaps during matrix assembly, and that the 5C5 epitope may serve as a marker for assembled laminin-5r matrix.
v-Src induces constitutive macropinocytosis in rat fibroblasts
Authors: Veithen Alex , Cupers Philippe , Baudhuin Pierre , Courtoy Pierre J.
Published: April 2021

The role of v-Src as regulator of fluid-phase pinocytosis was investigated in Rat-1 cells expressing a stable (Rat-1/BB16) or a thermosensitive (Rat-1/tsLA29) v-Src protein. In the second cell line, this protein is inactive when cells are cultured at 40°C but recovers its tyrosine kinase activity upon transfer to 34°C, resulting into a transformed phenotype. The rate of fluid-phase pinocytosis of the tracer horseradish peroxidase was 2-fold higher in v-Src-transformed fibroblasts (Rat-1/BB16, Rat-1/tsLA29 cultured at 34°C) as compared to nontransformed cells (Rat-1, Rat-1/tsLA29 kept at 40°C). In contrast, receptor-mediated endocytosis of transferrin was poorly affected, suggesting that structures distinct from clathrin-coated pits are involved in pinocytosis stimulation. By light and electron microscopy, transformed cells frequently contained large peroxidase-labeled pinocytic vesicles located near to membrane ruffles, demonstrating that stimulation of pinocytosis corresponds to induction of constitutive macropinocytosis.

Stimulation of pinocytosis occurred more than 8 hours after transfer to the permissive temperature, whereas transfer to the non-permissive temperature partially reversed the stimulation within 2 hours. Protein synthesis inhibition for 6 hours abrogated pinocytosis stimulation in transformed cells, indicating that constitutive macropinocytosis induced by v-Src depends on continuous synthesis of a short-lived regulatory machinery.
Induction of constitutive melanogenesis in amelanotic mouse melanoma cells by transfection of the human melanocortin-1 receptor gene
Authors: Tapia Johanna Chluba-de , Bagutt Claudia , Cotti Renato , Eberle Alex N.
Published: April 2021

The human melanocortin-1 (MC1) receptor was stably expressed in the amelanotic mouse melanoma cell clone B16-G4F which does not express its own (mouse) MC1 receptor and hence is unresponsive to α melanocyte stimulating hormone (αMSH). From several stable transfectant cell lines expressing the human MC1 receptor in relatively high numbers, three melanin producing clones (G4F-12, 14, and 15) and one amelanotic clone (G4F-7) were further analyzed in competition binding experiments and in cAMP and melanin assays. The dissociation constants (KD) for [Nle4, D-Phe7]-αMSH in all four clones ranged from 0.187 to 0.705 nmol/l, thus corresponding to the KD observed with the different human melanoma cell lines so far studied. Intracellular cAMP content was 3-to 5-fold higher than that of control cells, and αMSH induced an additional 1.5-to 1.7-fold increase. G4F-15 cells secreted melanin into the medium whereas the other clones did not secrete melanin. The extent of melanin secretion was similar to that of fully αMSH-stimulated B16-F1 mouse melanoma cells but the onset of secretion was delayed. αMSH induced an additional dose-related increase (up to 1.3-fold) in melanin production which could be suppressed by the addition of specific αMSH antibodies without altering the constitutive part of melanogenesis. Human and mouse agouti proteins, which inhibit basal and αMSH-induced melanogenesis in B16-F1 cells, both reduced αMSH-induced melanin production in G4F-15 cells but did not affect the constitutive melanogenesis. These results indicate that human MC1 receptor expressed in mouse B16-G4F cells induces constitutive activation of the signalling pathway controlling melanogenesis, most likely by tightly coupling to Gsα, in a similar manner to that reported for constitutively active receptor mutants in other systems.
Identification of two distinctly localized mitochondrial creatine kinase isoenzymes in spermatozoa
Authors: Kaldis Philipp , Stolz Martin , Wyss Markus , Zanolla Else , Rothen-Rutishauser Barbara , Vorherr Thomas , Wallimann Theo
Published: April 2021

The creatine kinase (CK) isoenzyme system is essential for motility in rooster and sea urchin sperm. In the present study, biochemical characterization as well as immunofluorescence and confocal laser microscopy with highly specific antibodies against various chicken CK isoenzymes revealed that cytosolic brain-type CK isoenzyme (B-CK) is the only CK isoenzyme in rooster seminal plasma, while three isoenzymes, cytosolic B-CK, sarcomeric mitochondrial CK (Mib-CK), and a variant of ubiquitous Mi-CK (‘Mia-CK variant’), are found in rooster spermatozoa. These three isoenzymes are localized in different regions of the sperm cell. B-CK and Mib-CK were localized along the entire sperm tail and in the mitochondria-rich midpiece, respectively. The ‘Mia-CK variant’, on the other hand, was found predominantly at the head-midpiece boundary, in a non-uniform manner in the midpiece itself and, surprisingly, at the distal end of the sperm tail as well as at the acrosome. Several lines of evidence show that the ‘Mia-CK variant’ shares some characteristics with purified Mia-CK from chicken brain, but also displays distinctive features. This is the first evidence for two different Mi-CK isoenzymes occurring in one cell and, additionally, for the coexpression of Mib-CK and cytosolic brain-type B-CK in the same cell. The relevance of these findings for sperm physiology and energetics is discussed.
The internalization of a short acyl chain analogue of ganglioside GM1 in polarized neurons
Authors: Sofer Anat , Schwarzmann Güenter , Futerman Anthony H.
Published: April 2021

In order to study the endocytosis of membrane lipids during the development of neuronal polarity, we examined the internalization of a short acyl chain fluorescent derivative of ganglioside GM1, N-(6-(4-nitrobenz-2-oxa-1,3-diazole-7-yl)-aminohexanoyl)-GM1 (C6-NBD-GM1), in hippocampal neurons cultured at low density. C6-NBD-GM1 was internalized by temperatureand energy-dependent mechanisms, and after short times of incubation, accumulated in endosomes in the axon, cell body and dendrites of neurons maintained for up to 4-5 days in culture. C6-NBD-GM1 was subsequently transported in a retrograde direction to a pool of recycling endosomes in the cell body, with little transport to lysosomes, as indicated by the lack of degradation of C6-NBD-GM1 even after long times, and the re-appearance of intact C6-NBD-GM1at the cell surface after recycling; similarly, little degradation of C6-NBD-GM1 was detected in N18TG-2 neuroblastoma cells. In hippocampal neurons maintained for longer than 6 days in culture, there was little internalization of C6-NBD-GM1 along the length of axons, but the amount of endocytosis from dendrites was similar to that observed in younger neurons. These results demonstrate that gangliosides turnover rapidly in dendritic membranes at all stages of neuronal development, whereas ganglioside turnover in axons is much less rapid, at least in mature, polarized neurons.
The basolateral sorting signal of the polymeric immunoglobulin receptor contains two functional domains
Authors: Reich Vanda , Mostov Keith , Aroeti Benjamin
Published: April 2021

Basolateral sorting of the polymeric immunoglobulin receptor (pIgR) expressed in Madin-Darby canine kidney (MDCK) cells is mediated by a 17-residue sorting signal that resides in the cytoplasmic domain. We have recently analyzed the sequence requirements of the signal by alanine scanning mutagenesis. We found that basolateral sorting is mediated primarily by three amino acids: H656, R657 and V660. Individual mutations of each of these residues to Ala caused a substantial decrease in basolateral sorting and a corresponding increase in targeting to the apical surface. Structural analysis of 17-residue peptides corresponding to the signal revealed that V660 is in a β-turn (probably type I) secondary structure, and its mutation to Ala destabilized the turn. H656 and R657 were not part of the turn and substitution of Arg657 to Ala had no effect on the turn stability. These results suggested that the signal is comprised of two structurally distinct domains: a critical V660 in the context of the β-turn and an additional two residues (H656 and R657) that are not in the turn and probably are unimportant for its stability. Here we provide evidence suggesting that the two domains are distinguishable not only by their structure but also by their function. Basolateral targeting of pIgR mutants bearing Ala mutations at either 656 or 657 was not affected by treatment with brefeldin A (BFA), while basolateral targeting of pIgR containing an Ala substitution at position 660 was markedly and uniquely stimulated by BFA. Compared to single Ala substitutions, simultaneous mutations of H656 and R657 to Ala caused an additional minor effect on basolateral and apical sorting, whereas double mutations of V660 and either H656 or R657 resulted in a maximal decrease in basolateral targeting and corresponding increase in apical targeting. These results suggest the existence of two domains in the signal. When both domains are destroyed, basolateral targeting is maximally inhibited. The results also imply that V660 mediates basolateral sorting by a different mechanism from H656 and R657. We suggest that V660 and perhaps more generally the β-turn may interact with BFA-sensitive adaptor complexes.
Focal cellular origin and regulation of interstitial collagenase (matrix metalloproteinase-1) are related to menstrual breakdown in the human endometrium
Authors: Kokorine I. , Marbaix E. , Henriet P. , Okada Y. , Donnez J. , Eeckhout Y. , Courtoy P. J.
Published: April 2021

Recent studies suggest that interstitial collagenase (MMP-1) is an essential enzyme in the early events leading to menstruation. This study analyses its cellular origin, regulation and relation to extracellular matrix breakdown in the human endometrium, both in cultured and non-cultured samples. The source of MMP-1 was identified by in situ hybridization and by immunohistochemistry on serial sections. This was compared with the immunolocalization of other MMPs, steroid receptors, macrophages, and laminin. In non-cultured endometrium, MMP-1 was only expressed during the perimenstrual period. It was either restricted to superficial foci of stromal cells or extended towards the entire functional layer. MMP-1 expression remarkably correlated with matrix breakdown, as assessed by silver staining, and was prominent at the periphery of shedding fragments and along some arterioles. In cultured non-menstrual explants, MMP-1 expression was induced within two days after deprivation of sex steroids. Both in cultured and non-cultured samples, progesterone receptors were not detectable in epithelial cells at foci of MMP-1 expression. The same stromal cells could synthesize MMP-1, MMP-2 (gelatinase A) and MMP-3 (stromelysin-1), as well as laminin, and did not correspond to macrophages. In conclusion, MMP-1 is focally expressed in stromal cells of the functional layer of the endometrium, when and where steroid receptors disappear, and especially where tissue breakdown is prominent. These observations point to an essential role for MMP-1 in the early stages of menstruation.
Alterations in neural intermediate filament organization: Functional implications and the induction of pathological changes related to motor neuron disease
Authors: Straube-West Karen , Loomis Patricia A. , Opal Puneet , Goldman Robert D.
Published: April 2021

The properties regulating the supramolecular organization of neural intermediate filament (NIF) networks have been investigated in cultured dorsal root ganglion (DRG) neurons. The studies described take advantage of the ability of endogenous NIF to incorporate purified biotinylated neurofilament triplet (NFT) proteins, NF-L, NF-M and NF-H. When injected at concentrations of 0.8-1.0 mg/ml injection buffer, each of these proteins is incorporated without perturbing the endogenous NIF network. However, at progressively higher concentrations, NF-H induces the aggregation and accumulation of NIF in the cell body. Subsequent to the induction of these aggregates, numerous alterations in the cytoarchitecture of neurons can be detected. The latter occur in a temporal sequence which appears to begin with the fragmentation of the Golgi complex. At later times, accumulation of mitochondria within the proximal region of neurites, peripheralization of the nucleus, and a significant decrease in neurite caliber become obvious. After longer time periods, the NIF aggre gates are seen to react with an antibody which reveals abnormally phosphorylated NF-H. These observations demonstrate that an imbalance in the normal stoichiometric relationships among the NFT proteins rapidly alters the supramolecular organization of the NIF network. These changes most likely reflect the normal functions of neurofilaments in cell shape and the organization and cytoplasmic distribution of membranous organelles. Interestingly, virtually all of these changes closely resemble those which have been reported in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). These findings suggest that cultured neurons can be used as models for more precisely defining the relationships between the formation of N?F aggregates and the sequence of cytopathological events which typify neurodegenerative diseases.
An apoptotic endonuclease activated either by decreasing pH or by increasing calcium
Authors: Collins Mary K. L. , Furlong Isla J. , Malde Prupti , Ascaso Rosalia , Oliver Javier , Rivas Abelardo Lopez
Published: April 2021

DNA fragmentation in isolated nuclei from the murine IL3-dependent bone marrow cell line BAF3 could be stimulated either by decreasing pH below 6.5 or by adding μM calcium at neutral pH. An endonuclease which could also be stim-ulated either by a decrease in pH, to 6.5, or by the presence of μM calcium at neutral pH, was purified 104-fold from nuclei of BAF3 cells. Digestion of DNA with the purified enzyme resulted in 5′-terminal hydroxyl and 3′-terminal phosphate ends. These characteristics are distinct from those described for other mammalian endonucleases. The possible role of this enzyme in genome digestion during apoptosis is discussed.
A method of maintaining parenchymal cells from adult rat liver in vitro
Authors: Alwen J. , Gallhai-Atchard Jennifer J.
Published: April 2021

A method for preparing suspensions of adult rat hepatocytes suitable for maintenance in vitro is described. Cultures were established from the cell suspensions by the squash technique. Cells were examined by light and electron microscopy; histochemically for glycogen, bile, lipid and glucose-6-phosphatase; and by autoradiography for DNA, RNA and protein synthesis. Hepatocytes could be maintained in vitro for at least 3 days and began to aggregate after 1 day. Uridine and leucine were incorporated, but not thymidine. Cultures consisted mainly of hepatocytes, though reticulo-endothelial cells were sometimes present.
Helical filaments in the glial cells of the locust (Schistocerca gregaria)
Authors: Osborne M. P.
Published: April 2021

Organelles consisting of a spirally wound filament are present in the cytoplasm of glial cells in Schistocerca gregaria. The diameter of the helix is 32·5 ran with an average pitch length of 23·0 ran. The filament is comprised of only a single strand about 6·0 nm in diameter which appears to be made up of a chain of globular subunits. In order to distinguish between these helical structures and organelles made up of more than one strand, i.e. microtubules and microfilaments, it is suggested that they be termed ‘microhelices’.

The structural features of microtubules, microfilaments and microhelices are compared, and the possible functions of microhelices are discussed in the light of what is known about the physiological properties of microtubules.

It is pointed out that the microhelix is structurally very similar to the internal ribonucleo-protein component of the influenza virus, and that the possibility cannot be discounted that the microhelix might be a viral particle.
Acceleration of nuclear growth and advanced initiation of mitosis in u.v.-irradiated plasmodia of Physarum polycephalum
Authors: Telatnyk Maureen M. , Guttes E.
Published: April 2021

In the multinucleated plasmodia of Physarum polycephalum the intermitotic period lasts from 2·5–5 days on diluted (1:8) growth medium and 3 − 7 days on non-nutrient balanced salt solution. When such plasmodia were irradiated with u.v. light approx. 15 − 20 h after mitosis, the next mitosis was considerably advanced and the intermitotic period lasted only between 36 − 48 h. During this time, numerous nuclei became pyknotic and disappeared, while the others became larger than in unirradiated controls. It is proposed that the abbreviation of the inter-mitotic period preceding the first post-irradiation mitosis was caused by incorporation into the surviving nuclei of a substance(s) which was released from the degenerating nuclei.
Fine-structural studies on spontaneous and induced fusion of higher plant protoplasts
Authors: Withers Lyndsey A. , Cocking E. C.
Published: April 2021

During the treatment of some plant tissues with cell-wall-degrading enzymes adjacent cells within the tissue fuse forming large multinucleate protoplasts. These have been termed spon-taneous fusion bodies. The symplastic nature of plant tissues suggests that the retention of plasmodesmatal connexions might facilitate such spontaneous fusion. An electron-microscopic investigation of spontaneous fusion in tobacco-leaf and oat-root tissues has confirmed this suggestion. Enzymic degradation of the walls removes constrictions on the plasmodesmata, permitting their expansion, and as a result mixing of the cytoplasms of the fusing protoplasts can then occur.

The fine structure of plasmodesmata and their relationship to the endoplasmic reticulum can be more easily studied in plasmodesmata which are undergoing expansion. It has been observed that the tubule which passes through the plasmodesma is in continuity with the endoplasmic reticulum membranes at either end. Models for plasmodesmatal structure are discussed in the light of this observation.

The induced fusion of freely isolated protoplasts by sodium salts has been previously studied using the light microscope. Since it is difficult to follow the detailed mechanisms involved in the process, electron-microscopic methods have been employed in the present investigation. It appears that sodium nitrate first induces protoplast adhesion. This occasionally involves protrusions from the plasmalemma, not unlike microvilli. Following adhesion membrane fusion occurs, initially in localized regions, and then more generally. Eventually vacuolar fusion occurs facilitating complete cytoplasmic mixing.

These findings are compared with events occurring during animal cell fusion and are discussed in relationship to a recent theoretical model for membrane fusion.
The fine structure of the embryogenic callus of Ranunculus sceleratus l
Authors: Thomas E. , Konar R. N. , Street H. E.
Published: April 2021

Sections of callus of Ranunculus sceleratus reveal its organization into cellular aggregates, the superficial cells of which are highly cytoplasmic and the inner cells of which are larger, have less-dense cytoplasm and are more highly vacuolated. Expansion and ultimate death of some of the internal cells leads to cell separation and break up of the aggregates.

Many of the superficial cells are involved in the initiation of embryoids in the callus and they correspond in structure with the embryogenic cells of the seedling stem epidermis. The embryoids are retarded in their development in presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and embryoid development becomes more rapid and more prolific when cultures are transferred to a medium without 2,4-D.

The similarity between the embryogenic cells of the callus cultures and those of the seedling stem epidermis extends to cell size, nuclear size, degree of vacuolation, abundance of ribosomes and mitochondria, presence of amyloplasts and prominence of spherosomes. The various forms of spherosome are described and their possible function discussed. Amyloplasts differentiating into chloroplasts are observed in the more advanced embryoids.

There is evidence that embryoids can arise from single cells but it is uncertain whether all are of single-cell origin. The embryogenic cells are in protoplasmic continuity with surrounding cells when they embark upon embryogenesis.

Some of the superficial cells, also clearly undergoing active division, are of rather different structure; characteristically their nuclei show a high degree of chromatin condensation and their cytoplasm contains bundles of fibrous material. It is suggested that these cells do not function directly in embryogenesis.

The internal cells of the aggregates have a low density of ribosomes and very few ER profiles or normal mitochondria. Extremely elongated mitochondrial structures following the outline of the nucleus are observed in these cells.

An unidentified structure is frequently observed in cells in which cytoplasmic disorganization appears to be occurring.
Raphide crystal cell development in leaves ofPsychotria punctata(rubiaceae)
Authors: Horner H. T. , Whitmoyer R. E.
Published: April 2021

The distribution and development of raphide cry8tal cells in nodulated leaves of Psychotria punctata were studied by light and electron microscopy. Crystal cells in the leaf are oriented in various ways depending on whether they occur in the spongy or palisade parenchyma. Crystals are never found within the bacterial nodules and are not concentrated around them. Developing leaf crystal cells become larger than surrounding cells and have larger nuclei and nucleoli. Raphides develop within membrane chambers in the large central vacuole in association with membrane complexes, vesicles and tubules, the latter measuring 10–13 nm in diameter. Certain cytoplasmic organelles, the plasmalemma, and a cytoplasmic vacuolar channel complex also appear to be associated with crystal development. These results are compared with other recent investigations dealing with calcium oxalate crystals in higher plants.
Variable amounts of dna related to the size of chloroplasts: IV. three-dimensional arrangement of dna in fully differentiated chloroplasts of Beta vulgaris L
Authors: Kowallik K. V. , Herrmann R. G.
Published: April 2021

Glutaraldehyde-fixed strips of fully differentiated leaves of Beta vulgaris L. were treated with proteases. With this method it is possible to display DNA-regions in chloroplasts selectively and to reconstruct them from serial sections.

As in young plastids, the DNA in mature plastids is also distributed within several regions which are separated rather clearly by thylakoids. The number of such regions depends upon the size of the organelle and thus upon its developmental state. The shape of the regions is, in contrast to less-differentiated plastids, mostly elongated. The individual regions seem to contain unequal quantities of DNA-fibrils.

A comparison of our ultrastructural results and biochemical data (DNA-amounts per plastid depending on the size of the organelle; kinetic complexity of chloroplast-DNA of Beta) as well as a comparison of the chloroplast with similar prokaryotic systems (as in bacteria, blue-green algae, and mitochondria) leads to the suggestion that each DNA-containing region can be regarded as a single nucleoid. In addition, each nucleoid already contains several (on average, 4–8) genetic units. Thus the chloroplast of Beta seems genetically polyvalent in at least 2 respects: (i) it is of polyenergidic organization, and (ii) the individual nucleoids can be polyploid to varying degrees.
Further experiments on the role of the nucleolus in the expression of structural genes
Authors: Deák I. , Sidebottom E. , Harris H.
Published: April 2021

When a chick erythrocyte nucleus is introduced into the cytoplasm of a mouse cell it under-goes reactivation and eventually determines the synthesis of chick-specific proteins in the cytoplasm of the heterokaryon. It has previously been shown that there is a close temporal correlation between the onset of synthesis of the chick-specific proteins and the appearance of nucleoli in the erythrocyte nuclei. We now show that the synthesis of chick-specific proteins in the heterokaryon decays if the nucleolus in the reactivated erythrocyte nucleus is irradiated with an ultraviolet microbeam. The synthesis of chick-specific proteins does not, however, decay if only one of two nucleoli in the erythrocyte nucleus or if extranucleolar areas of the nucleus are irradiated. These observations confirm that some function located at or near the nucleolus is essential for the full expression of structural genes.
The effect of retinol and of retinol binding protein on embryonic skeletal tissue in organ culture
Authors: Dingle J. T. , Fell Honor B. , Goodman D. S.
Published: April 2021

Retinol, bound non-specifically to serum proteins in organ culture medium, caused the degradation of the extracellular matrix of chick limb-bone rudiments. The loss of polymeric material was demonstrated histologically and measured chemically.

Retinol, at the same concentration, bound specifically to retinol-binding protein, produced no significant effect. The addition of prealbumin to this system was also without discernible effect.

The addition of apo-RBP to medium containing free retinol prevented the normal hyper-vitaminous A effects. The results indicate that the retinol-RBP complex has a high stability, sufficient to prevent the uptake, by the plasma or lysosomal membranes, of the amount of retinol needed for physical changes to occur.

It is suggested that retinol-binding protein may serve to transport retinol to selected sites, whilst protecting biological membranes against a general non-specific effect of the vitamin’s surface active properties.