bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of madin-darby canine kidney (mdck) cells
Authors: Ojakian George K. , Schwimmer Randi 
Published: April 2021

ABSTRACT
Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by ∼33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.
A novel vesicle-associated protein (vap-1) in sea urchin eggs containing multiple rna-binding consensus sequences
Authors: Barton N. R. , Bonder E. M. , Fishkind D. J. , Warren R. H. , Pratt M. M. 
Published: April 2021

ABSTRACT
We have identified a novel high molecular weight, vesicle-associated protein (VAP-1) in the eggs of the sea urchin Strongylocentrotuspurpuratus. Biochemical fractionation and immunofluorescence analysis of unfertilized eggs indicate that VAP-1 is a peripheral membrane protein associated with microsomal membrane fractions. Sequence analysis of partial VAP-1 cDNA clones reveals that the protein contains at least four RNA-bind-ing consensus sequences. The RNA-binding sequences are separated by several glycine rich domains and this organization, RNA-binding domains separated by glycine rich sequences, is common to several RNA-binding proteins including the heterogeneous ribonuclear protein A1 and nucleolin. The characteristics of VAP-1 suggest that the protein may function as a multidomain RNA-binding protein. The possibility that VAP-1 may play a role in nuclear RNA processing is also discussed.
A block in degradation of mhc class ii-associated invariant chain correlates with a reduction in transport from endosome carrier vesicles to the prelysosome compartment
Authors: Zachgo Sabine , Dobberstein Bernhard , Griffiths Gareth 
Published: April 2021

ABSTRACT
Invariant chain (li) associated with MHC class II molecule is processed proteolytically via several distinct intermediates during its intracellular transport through endosomal compartments. Leupeptin added to the culture medium blocks processing of Ii, prevents its dissociation from the class II molecules and leads to an intracellular accumulation of a 22 kDa intermediate form of Ii. We show here that leupeptin has a very general effect on protein transport in the endocytic pathway. When added to Mel Juso cells leupeptin reduces the transport of endocytosed material from multivesicular body-like, endosome carrier vesicles (ECV) to the prelysosomal compartment (late endosome) and leads to a concomitant increase in the number of ECV. Our results argue that one effect of leupeptin, related to antigen processing and presentation, is to block transport of antigen and/or MHC class II molecules to prelysosomal compartments.
The relative positions of condensed chromosomes are maintained between divisions in the epidermis of Calpodes ethlius
Authors: Jeun Gwendolyn , Locke Michael 
Published: April 2021

ABSTRACT
The larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells are twins that remain connected by residual midbodies between mitoses. Twins resemble one another more than their other neighbours in such structural features as the shape and number of nucleolar particles and the number of actin bundles. We have now found that they also resemble one another in the position of the condensed chromosomes that occur in female cells. Female lepidopteran cells contain one or more particles of condensed chromatin, depending on their ploidy. In the epidermis, nuclei with two condensed chromosomes are found in pairs and are separated by the same distances. However, clones of cells with multiple condensed chromosomes are not all alike, suggesting that chromosomes are repositioned at mitosis. Separation distances between chromosomes remain the same between but not through cell divisions, suggesting that determinants for nuclear structure are conserved through interphase and relaxed at mitosis. Although the condensed chromosomes of sibling nuclei resemble one another in their separation, they differ in their orientation, as would be expected if whole nuclei rotate in the plane of the epithelium.
Proteases are not involved in the membrane fusion events of the lysolecithin-mediated guinea pig sperm acrosome reaction
Authors: Flaherty Sean P. , Swann Nicholas J. 
Published: April 2021

ABSTRACT
The guinea pig sperm acrosome reaction is characterized by a complex temporal and structural pattern of membrane fusions. In this study, we have used specific protease inhibitors to determine if proteases regulate this pattern of membrane fusions during the lysolecithin-mediated guinea pig sperm acrosome reaction. Inhibitors were chosen so as to cover a wide range of different types of proteases, and all were used at the highest concentration that did not adversely affect sperm motility. Of the eight inhibitors tested, leupeptin, soya bean trypsin inhibitor (SBTI), p-aminobenzamidine (pAB) and nitrophenyl p′ -guanidino benzoate (NPGB) inhibited completion of the acrosome reaction, while diethylenetriaminepentaacetic acid (DTPA), phosphoramidon, bestatin and pepstatin had no effect. Sperm that had been acrosome-reacted in the presence of each inhibitor were examined by transmission electron microscopy to assess whether the inhibitors altered the pattern of membrane fusions during the acrosome reaction. DTPA, phosphoramidon, bestatin and pepstatin had no effect on membrane fusion or matrix dispersal. Serine protease inhibitors such as leupeptin, SBTI, pAB and NPGB prevented complete dispersal of the acrosomal matrix and completion of the acrosome reaction, but did not alter the temporal sequence or structural pattern of membrane fusions. The undispersed matrix was present along the dorsal and ventral aspects of the apical segment and throughout the principal segment. We conclude that proteases are not involved in regulating the temporal and structural pattern of membrane fusions which occurs during the lysolecithin-mediated acrosome reaction of guinea pig sperm.
Platelet activating factor modulates signal transduction in Dictyostelium
Authors: Sordano Concetta , Cristino Elena , Bussolino Federico , Wurster Bernd , Bozzaro Salvatore 
Published: April 2021

ABSTRACT
During development, Dictyostelium discoideum cells produce platelet activating factor (PAF). When cells are stimulated with external cAMP pulses, PAF is transiently synthesized. To determine whether PAF is involved in signal transduction, we have tested the effect of PAF on some cellular responses which are regulated by cAMP, such as spontaneous light-scattering oscillations of suspended cells, cAMP relay, transient increases of cGMP level, and extracellular calcium uptake.
Our results show that PAF specifically interferes with spontaneous spike-shaped oscillations, without affecting sinusoidal ones. PAF increases the amplitude of a spike, but has no effects on its phase or frequency. When cells fail to oscillate spontaneously, PAF does not induce spikes; however, if administered together with cAMP, it amplifies the light-scattering response to cAMP. Amplification of light-scattering changes is accompanied by a threefold increase in the concentration levels of both cellular cAMP and cGMP. Extracellular Ca2 uptake is also stimulated by PAF. This latter response is independent of endogenous or exogenously added cAMP. All these effects are specific for the naturally occurring Renantiomer of PAF, the S-enantiomer and lyso-PAF being inactive. These results suggest that PAF modulates signal transduction in Dictyostelium, probably by interacting with an intracellular acceptor, which is involved in the pathways regulating membrane Ca2 +channels, adenylate and guanylate cyclase.
Analysis of conserved binding proteins for nuclear localization sequences
Authors: Stochaj Ursula , Bossie Mark A. , Zee Karen van , Whalen Anne M. , Silver Pamela A. 
Published: April 2021

ABSTRACT
Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex. It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore. These NLS-receptors could facilitate an early step of nuclear protein import, i.e. targeting and binding of nuclear proteins at the nuclear pore. We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms. Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells. Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores. One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein. Using isolated yeast nuclei we demonstrate that the antiidiotype antibodies compete for binding of nuclear proteins in vitro.
We show that the yeast mutant npl3, which is defective in nuclear protein localization, has an altered distribution of antigens recognized by these anti-idiotype antibodies, at the semi-permissive temperature. Our results suggest that a set of proteins common to various eukaryotes recognizes nuclear localization sequences.
F-actin domains in the syncytial blastoderm of the dipteran Ceratitis capitata
Authors: Callaini Giuliano , Dallai Romano , Riparbelli Maria Giovanna 
Published: April 2021

ABSTRACT
Laser scanning confocal microscopy on rhodamine-phalloidin-treated syncytial embryos of the dipteran Ceratitis capitata allowed us to recognize four different kinds of actin filament distribution in close spatial proximity. One domain is represented by microfilaments localized in the plasma membrane within the microprojections and membrane infoldings. At a slightly lower focal level, rhodamine-phalloidin labelling is concentrated in small irregular aggregates, which are localized around the dividing nuclei. Our results indicate that the organization of the actin aggregates follows that of the microtubules of the mitotic apparatus and suggest that the dynamic reorganization of these structures during mitosis may be microtubule-dependent. A threedimensional network of thin actin filaments fills the whole periplasm and links the spindles together. A fourth actin domain is localized at the poles of the spindles in correspondence with the centrosomal region. The complex network of cortical filament bundles described in the present study may represent the ultrastructural basis of the tension leading to segregation of daughter nuclei at late telophase and to their lateral migration along the embryo surface.
Deregulation of cyclins D1 and E and suppression of cdk2 and cdk4 in senescent human fibroblasts
Authors: Lucibello Frances C. , Sewing Andreas , Brüsselbach Sabine , Bürger Christiane , Müller Rolf 
Published: April 2021

ABSTRACT
The state of cellular senescence is characterised by an irreversible arrest in the G1 phase of the cell cycle. It has previously been shown that three cell cycle genes, cyclin A, cyclin B and cdc2, are not expressed in senes-cent human fibroblasts. All three gene products have functions after S-phase entry, so that their suppression cannot explain the irreversible G1 arrest. Here, we report that the abundance of transcripts from two other cell cycle genes, cdk2 and cdk4, thought to act during G1 S progression, is significantly diminished in senes-cent cells of the diploid human fibroblast line WI-38. Surprisingly, two other cyclins, D1 and E, behave in a completely different way, in that their expression is ele-vated in senescent cells, especially under conditions of serum starvation. Both the synthesis and the steady-state level of cyclin D1 protein were also found to be markedly higher in senescent cells (3-to 6-fold). Cyclins D1 and E are thus the first genes shown to be overex-pressed or deregulated in senescent cells. It is tempting to speculate that this deregulation may be due to the absence, in senescent cells, of a regulatory loop that would normally control their expression. This is sup-ported by our finding that cyclin E-associated kinase activity in senescent cells is reduced approx. 14-fold. Our data also suggest that the deregulated expression of cyclin D1 and E is not sufficient to drive senescent cells into DNA replication.
γ-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells
Authors: Julian M. , Tollon Y. , Lajoie-Mazenc I. , Moisand A. , Mazarguil H. , Puget A. , Wright M. 
Published: April 2021

ABSTRACT
Animal cells undergoing cytokinesis form an inter-cel-lular bridge containing two bundles of microtubules interdigitated at their plus ends, which constitute the midbody. Polyclonal antibodies raised against three specific amino acid sequences of γ-tubulin (EEFATEGGDRKDV, NIIQGEADPTDVHKSL and EYHAATRPDYISWGTQEQ) specifically stained the centrosome in interphase, the spindle poles in all stages of mitosis, and the extremities of the midbody in mam-malian cells (Potorous, human, Chinese hamster, mouse). This staining was prevented by the corre-sponding peptides, by Xenopus γ-tubulin, but was not modified by purified αγ-tubulin heterodimer. An iden-tical staining was obtained with affinity-purified anti-bodies against the carboxyl-terminal amino acid sequence of human γ-tubulin. No γ-tubulin could be detected in the interzone during anaphase and early telophase. Material containing γ-tubulin first appeared in the two daughter cells on each side of the division plane in late telophase, and accumulated transiently at the minus ends of the two microtubule bundles consti-tuting the midbody for one hour after metaphase. Micro-injection of γ-tubulin antibodies into anaphase cells prevented the subsequent formation of the micro-tubule bundles between the two daughter cells. In con-trast with previous views, these observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centres, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells.
Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes
Authors: Zambruno Giovanna , Marchisio Pier Carlo , Melchiori Antonella , Bondanza Sergio , Cancedda Ranieri , Luca Michele De 
Published: April 2021

ABSTRACT
Integrin receptors of human melanocytes in vivo and of melanocytes isolated and cultured from in vitro recon-stituted normal human epidermis were investigated. Melanocytes were studied by high-resolution immuno-cytochemistry of in situ epidermis and were found to expose only the integrin subunits α3, α6, αv and β1 on their plasma membrane surface. Instead, cultured normal melanocytes expressed α3β1, α5β1, α6β1 and αvβ3, which were immunoprecipitated from both meta-bolically and surface-labeled cells. Betα1 integrins were diffused on the adhesion surface, while αvβ3 was clus-tered in focal contacts both in control cells and upon dendrite induction with phorbol 12-myristate 13-acetate (PMA). The functional roles of integrins were studied in vitro by cell adhesion, spreading and migration assays. The sum of the data indicated that, in normal human melanocytes: (i) adhesion to defined substrata is mainly mediated by specific β1 integrins; (ii) spreading is mainly modulated by αvβ3; (iii) the β1 and β3 het-erodimers cooperate in regulating migration. The in vitro expression of two integrins (αvβ3 and α5β1) that are not exposed in situ, and their role in the spreading and migratory properties of melanocytes, strongly suggest that they are involved in regenerating a nor-mally pigmented epidermis during wound healing by controlling melanocyte spreading and migration over a provisional matrix. Tumor promoters, such as PMA, selectively increased the expression of α3β1. We suggest that this integrin might be involved in melanocyte migration on the newly formed basement membrane during wound healing as well as in intercellular recognition of adjacent keratinocytes.
Cyclic AMP- and Ins(1,4,5)P3-induced ca2+ fluxes in permeabilised cells of Dictyostelium discoideum: cGMP regulates ca2+ entry across the plasma membrane
Authors: Flaadt Heidrun , Jaworski Elke , Schlatterer Christina , Malchow Dieter 
Published: April 2021

ABSTRACT
Transduction of chemotactic signals in Dictyostelium dis -coideum apparently involves a precise regulation of the cytosolic ca2+ concentration. Cyclic AMP stimulation causes ca2+ influx followed by ca2+ extrusion, the mag-nitude of extrusion depending on the state of differen-tiation. Here, we show that the cAMP receptor controls ca2+ influx both at the level of entry across the plasma membrane and at the level of transport into ca2+-sequestering organelles. The use of permeabilised cells allowed us to discriminate between both fluxes. Perme-abilised cells still showed the cAMP-induced ca2+ uptake. The flux across the plasma membrane was more sensitive to Bα2+ and Mn2+, respectively, than ca2+ sequestration. We have shown previously, using stmF mutants, that cGMP regulates ca2+ influx. We confirmed this result with the membrane-permeant cGMP-analogue, Sp-8-Br-cGMPS, which enhanced the cAMP-induced ca2+ influx in intact cells but not the uptake in permeabilised cells, indicating that cGMP regulates ca2+ influx across the plasma membrane. Occasionally, a fast transient ca2+ efflux, preceding the influx, occurred in intact cells. A small cAMP-induced ca2+ release was also found in permeabilised cells. A simi-larly sized ca2+ release was elicited by Ins(1,4,5)P3 and could be substituted for by GTP or GTPyS. This result suggests that rapid ca2+ release can be mediated by Ins(1,4,5)P3.
Non-enzymatic access to the plasma membrane of Medicago root hairs by laser microsurgery
Authors: Kurkdjian Armen , Leitz Guenther , Manigault Pierre , Harim Abdellah , Greulich Karl Otto 
Published: April 2021

ABSTRACT
Using UV laser microsurgery, the cell walls of root hairs from Medicago sativa (alfalfa) were perforated under plasmolysing conditions, giving direct access to the plasma membrane without enzyme treatment. The opening in the cell wall of a few m in diameter results in immediate movement of the protoplasm and partial or complete extrusion of the cell contents. The move-ment of the protoplasm is retarded by increases in cal-cium concentration. The calcium-dependency of the movement of the protoplasm allows us to obtain pref-erentially the extrusion of protoplasm, or to gain access to a small area of plasma membrane in situ. The com-plete protoplasm can be expelled, to form a protoplast. Fluorescein diacetate staining indicated esterase activity and membrane integrity of the protoplasts. Microscopic examination revealed organelle movement and the pres-ence of a nucleus. The plasma membrane was free from cell wall fragments, as shown by Tinopal staining. Con-ditions for obtaining plasmolysis without disturbing the physiology of the root hairs too much were achieved by slow, stepwise and reversible plasmolysis. Cytoplasmic streaming in root hairs was maintained during plas-molysis and laser microperforation. This laser technique should be suitable for the performance of electrophysi-ological studies using the patch-clamp technique on plasma membrane from non-enzyme-treated cells.
Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts
Authors: Shankar Geetha , Davison Ian , Helfrich Miep H. , Mason William T. , Horton Michael A. 
Published: April 2021

ABSTRACT
Cell-matrix interactions have been shown to play an important role in regulating cell function and behav-iour. In bone, where calcified matrix formation and resorption events are required to be in dynamic equi-librium, regulation of adhesive interactions between bone cells and their matrix is critical. The present study focuses on the osteoclast, the bone resorbing cell, as well as integrins, which are cell surface adhesion receptors that mediate osteoclast attachment to bone matrix. In osteoclasts, the most abundant integrin receptor is the vitronectin receptor (VNR, αv3). The objective of the study was to investigate changes in intracellular cal-cium, a regulator of osteoclast function, following addition of peptides that bind integrins, in particular the αv3 form of the vitronectin receptor (VNR), which is highly expressed in osteoclasts.
The study demonstrated a unique spatial localisation of the calcium signal in response to cell membrane receptor occupancy by integrin ligands in rat osteo-clasts. Addition of peptides with the Arg-Gly-Asp (RGD) sequence such as BSP-IIA, GRGDSP and GRGDS to rat osteoclasts evoked an immediate increase in free calcium ion concentration [Ca2+]i, localised to the nuclei and to the thin cytoplasmic skirt. These responses were inhibited by F11, a monoclonal antibody to the rat integrin 3 chain, as well as echistatin, a snake venom shown to colocalise with the αv chain in osteoclasts, suggesting that the calcium signal is mediated by the αv3 form of VNR. In contrast, a uniform increase in [Ca2+]i through-out the osteoclast was observed with the calcium-regu-lating hormone calcitonin, as a consequence of calcium entry from extracellular sources. Addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a non-nuclear calcium ATPase inhibitor, resulted in a calcium signal with spatial characteristics distinct from that evoked by calcitonin or RGD-containing peptides. Thus, an increase in intranuclear calcium to the exclusion of a concomitant rise in cytoplasmic [Ca2+]i was restricted to responses induced by RGD-containing peptides.
The present findings demonstrate that, in rat osteo-clasts, there is a signalling pathway linked to the inte-grin VNR, which can rapidly modulate the concentration of nuclear [Ca2+]i, which in turn may regulate nuclear calcium-dependent processes.