bluuebunny/crossref_metadata_2025 huggingface dataset:
Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii
Authors: Morrissette Naomi S. , Murray John M. , Roos David S.
Published: April 2021

Application of Fourier analysis techniques to images of isolated, frozen-hydrated subpellicular microtubules from the protozoan parasite Toxoplasma gondii demonstrates a distinctive 32 nm periodicity along the length of the microtubules. A 32 nm longitudinal repeat is also observed in the double rows of intramembranous particles seen in freezefracture images of the parasite’s pellicle; these rows are thought to overlie the subpellicular microtubules. Remarkably, the 32 nm intramembranous particle periodicity is carried over laterally to the single rows of particles that lie between the microtubule-associated double rows. This creates a two-dimensional particle lattice, with the second dimension at an angle of ∼75° to the longitudinal rows (depending on position along the length of the parasite). Drugs that disrupt known cytoskeletal components fail to destroy the integrity of the particle lattice. This intramembranous particle organization suggests the existence of multiple cytoskeletal filaments of unknown identity. Filaments associated with the particle lattice provide a possible mechanism for motility and shape change in Toxoplasma: distortion of the lattice may mediate the twirling motility seen upon host-cell lysis, and morphological changes observed during invasion.
SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface
Authors: Marsh Mark , Bron Romke
Published: April 2021

Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acidinduced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentallyinduced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus.

These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha- and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.
Plasma membrane targetting, vesicular budding and release of galectin 3 from the cytoplasm of mammalian cells during secretion
Authors: Mehul Bruno , Hughes R. Colin
Published: April 2021

Galectin 3, a 30 kDa galactoside-binding protein distributed widely in epithelial and immune cells, contains no signal sequence and is externalized by a mechanism independent of the endoplasmic reticulum (ER)-Golgi complex. We show here that hamster galectin 3 overexpressed in transfected cos-7 cells is secreted at a very low rate. A chimaera of galectin 3 fused to the N-terminal acylation sequence of protein tyrosine kinase p56lck, Nt-p56lck-galectin 3, which is myristoylated and palmitoylated and rapidly transported to plasma membrane domains, is efficiently released from transfected cells indicating that movement of cytoplasmic galectin 3 to plasma membrane domains is a rate limiting step in lectin secretion. N-terminal acylation is not sufficient for protein secretion since p56lck and the chimaera Nt-p56lck-CAT are not secreted from transfected cells. The amino-terminal half of galectin 3 is sufficient to direct export of a chimaeric CAT protein indicating that part of the signal for plasma membrane translocation lies in the N-terminal domains of the lectin. Immunofluorescence studies show that Nt-p56lck-galectin 3 aggregates underneath the plasma membrane and is released by membrane blebbing. Vesicles of low buoyant density isolated from conditioned medium are enriched in galectin 3. The lectin is initially protected from exogenous collagenase but is later released in soluble protease-sensitive form from the lectin-loaded vesicles. Using murine macrophages, which secrete their endogenous galectin 3 at a moderate rate especially in the presence of Ca2+-ionophores, we were also able to trap a galectin 3-loaded vesicular fraction which was released into the culture supernatant.
Localization of five annexins in J774 macrophages and on isolated phagosomes
Authors: Diakonova Maria , Gerke Volker , Ernst Joel , Liautard Jean-Pierre , van der Vusse Ger , Griffiths Gareth
Published: April 2021

Annexins are a family of structurally related proteins which bind phospholipids in a calcium-dependent manner. Although the precise functions of annexins are unknown, there is an accumulating set of data arguing for a role for some of them in vesicular transport and, specifically, in membrane-membrane or membrane-cytoskeletal interactions during these processes. Here we describe our qualitative and quantitative analysis of the localization of annexins I-V in J774 macrophages that had internalized latex beads, both with and without IgG opsonization. Our results show that whereas all these annexins are present on both the plasma membrane and on phagosomes, the localization on other organelles differs. Annexins I, II, III and V were detected on early endosomes, while only annexin V was seen on late endocytic organelles and mitochondria. Annexins I and II distributed along the plasma membrane non-uniformly and co-localized with F-actin at the sites of membrane protrusions. We also investigated by western blot analysis the association of annexins with purified phagosomes isolated at different time-points after latex bead internalization. While the amounts of annexins I, II, III and V associated with phagosomes were similar at all times after their formation, the level of annexin IV was significantly higher on older phagosomes. Whereas annexins I, II, IV and V could be removed from phagosome membranes with a Ca2+ chelator they remained membrane bound under low calcium conditions. In contrast, annexin III was removed under these conditions and needed a relatively high Ca2+ concentration to remain phagosome bound. Because of their purity and ease of preparation we suggest that phagosomes are a powerful system to study the potential role of annexins in membrane traffic.
Fibroblast growth factor receptor signalling has a role in lobuloalveolar development of the mammary gland
Authors: Jackson David , Bresnick Janine , Rosewell Ian , Crafton Tracy , Poulsom Richard , Stamp Gordon , Dickson Clive
Published: April 2021

We have used the mouse mammary tumor virus promoter to express two dominant negative (DN) fibroblast growth factor receptor (FGFR) isoforms in the mammary epithelium of transgenic mice. While expression of DN-FGFR1(IIIc) showed no discernible phenotype, a similar kinase negative form of FGFR2(IIIb) caused a marked impairment of lobuloalveolar development. The growth retardation was apparent by mid-pregnancy and persisted in the post-partum glands. Despite the substantial underdevelopment of the mammary gland there was a measurable lactational response, but it was insufficient to properly sustain the new-born pups. These findings demonstrate that fibroblast growth factor signalling is necessary for pregnancy dependent lobuloalveolar development of the mammary gland.
Differentiation of embryonic stem cells into adipocytes in vitro
Authors: Dani C. , Smith A. G. , Dessolin S. , Leroy P. , Staccini L. , Villageois P. , Darimont C. , Ailhaud G.
Published: April 2021

Embryonic stem cells, derived from the inner cell mass of murine blastocysts, can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes, such as peroxisome proliferator-activated receptors γ and δ and CCAAT/enhancer binding protein β, during differentiation of retinoic acidtreated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes.
Phosphorylation regulates the assembly of NuMA in a mammalian mitotic extract
Authors: Saredi Alejandro , Howard Louisa , Compton Duane A.
Published: April 2021

NuMA is a 236 kDa nuclear protein that is required for the organization of the mitotic spindle. To determine how NuMA redistributes in the cell during mitosis, we have examined the behavior of NuMA in a mammalian mitotic extract under conditions conducive to the reassembly of interphase nuclei. NuMA is a soluble protein in mitotic extracts prepared from synchronized cultured cells, but forms insoluble structures when the extract becomes nonmitotic (as judged by the inactivation of cdc2/cyclin B kinase and the disappearance of mpm-2-reactive antigens). These NuMA-containing structures are irregularly shaped particles of 1-2 μm in diameter and their assembly is specific because other nuclear components such as the lamins remain soluble in the extract under these conditions. NuMA is dephosphorylated during this assembly process, and the assembly of these NuMA-containing structures is catalyzed by protein dephosphorylation because protein kinase inhibitors enhance their formation and protein phosphatase inhibitors block their formation. Finally, immunodepletion demonstrates that NuMA is an essential structural component of these insoluble particles, and electron microscopy shows that the particles are composed of a complex interconnected network of foci. These results demonstrate that phosphorylation regulates the solubility of NuMA in a mammalian mitotic extract, and the spontaneous assembly of NuMA into extensive structures upon dephosphorylation supports the conclusion that NuMA serves a structural function.
Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-β
Authors: Fridell Robert A. , Truant Ray , Thorne Leigh , Benson R. Edward , Cullen Bryan R.
Published: April 2021

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the welldefined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-β, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.
Calcium ion dependency and the role of inositol phosphates in melatonin-induced encystment of dinoflagellates
Authors: Tsim Siu-Tai , Wong Joseph T. Y. , Wong Yung H.
Published: April 2021

The unicellular eukaryotic dinoflagellates shed their flagella and form a new pellicle cyst wall in response to environmental stress. This encystment process can also be induced by indoleamines such as melatonin and 5-methoxytryptamine. To decipher the complex signaling events which lead to encystment, we have investigated the functional roles of Ca2+ and inositol phosphates in indoleamine-induced encystment of the dinoflagellates Alexandrium catenella and Crypthecodinium cohnii. Pretreatment with EGTA, but not with EDTA, effectively blocked the indoleamine-induced encystment of A. catenella in a dose-dependent manner. Conversely, agents that facilitate the influx of Ca2+ (Bay K 8644, A23187 and ionomycin) dose-dependently induced encystment of A. catenella. Endoplasmic Ca2+-ATPase inhibitors such as thapsigargin and the peptide toxin melittin also induced encystment of A. catenella. These results suggest that an elevation of intracellular [Ca2+] may be involved in the encystment response. In terms of the regulation of phospholipase C, melatonin doseand time-dependently stimulated the formation of inositol phosphates in C. cohnii. The rank order of potency for several indoleamines to stimulate inositol phosphates formation was 2-iodomelatonin > 5-methoxytryptamine ≥ melatonin >> N-acetylserotonin > 5-hydroxytryptamine. This rank order was the same as for the indoleamine-induced encystment of C. cohnii as previously reported. Our results indicate that indoleamineinduced activation of phospholipase C and elevation of intracellular [Ca2+] may be proximal steps in the signal transduction pathway leading to encystment in dinoflagellates. Moreover, this is the first demonstration of the possible involvement of Ca2+ and inositol phosphates as second messengers in dinoflagellates.
NPXY motifs control the recruitment of the α5β1 integrin in focal adhesions independently of the association of talin with the β1 chain
Authors: Vignoud Lucile , Albigès-Rizo Corinne , Frachet Philippe , Block Marc R.
Published: April 2021

With the exception of the divergent β4 and β8 chains, the integrin β subunit cytoplasmic domains are short and highly conserved sequences. Consensus motifs are found among the different cytoplasmic β chains. Experiments using chimeric receptors demonstrated that the 47 amino acids of the β1 subunit cytoplasmic domain contain sufficient information to target integrins to adhesion plaques. Three clusters of amino acids, named cyto-1, cyto-2 and cyto-3, seem to contribute to this localization. Cyto-2 and cyto-3 exhibit NPXY motifs. At present, the exact function of these motifs remains unknown but it is likely that these sequences are involved in proteinprotein interactions. Although NPXY motifs often act as internalization signals at the cytoplasmic tail of membrane receptors, our previous results showed that the two NPXY motifs are not responsible for the α5β1 integrin endocytosis. Herein, we address the question of the role of the two highly conserved NPXY motifs found in the β1 cytoplasmic domain, and which correspond to the conserved domains cyto-2 and cyto-3. We demonstrate that, within the integrin β1 cytoplasmic tail, the two NPXY motifs are required for the recruitment of the integrin in focal adhesions. In addition, our results indicate that these two motifs control but do not belong to the talin-binding sites. Finally, the analysis of the phenotypes of NPXY mutants reveals that the interaction of talin with the β1 cytosolic domain is not sufficient to target the integrins to focal adhesions.
Modulation of protein synthesis relative to DNA synthesis alters the timing of differentiation in the protozoan parasite Theileria annulata
Authors: Shiels Brian , Aslam Nasreen , McKellar Sue , Smyth Audra , Kinnaird Jane
Published: April 2021

The control of differentiation through time is critical for the correct ordering of sequential developmental events. A timing mechanism based on the number of mitotic divisions has been proposed for both higher eukaryote and protozoan parasite cellular differentiation. However, the mitotic clock model has not been validated by experiments which altered the proliferation rate of cells in vitro. This study has used the drugs aphidicolin and oxytetracycline to investigate the modulation of differentiation in the protozoan parasite Theileria annulata. The results showed that the timing of macroschizont to merozoite differentiation correlated with expression levels of a merozoite surface antigen during the reversible phase of the differentiation process. In addition, analysis of the effect of the drugs and elevation of culture temperature indicated that altered timing of differentiation was associated with changes to the rate of protein synthesis relative to DNA synthesis. From these results we postulate that the differentiation clock runs on the basis of a progressive elevation of a regulator(s) of merozoite gene expression to a quantitative commitment threshold. We also propose that this mechanism of timing can be corrupted by modulation of the proliferation potential (DNA synthesis) and/or growth potential (factor production) of the cell. The relevance of this model to differentiation in vivo and to other eukaryotic systems is discussed.
Leukosialin (CD43, sialophorin) redistribution in uropods of polarized neutrophils is induced by CD43 cross-linking by antibodies, by colchicine or by chemotactic peptides
Authors: Seveau Stéphanie , Lopez Sandra , Lesavre Philippe , Guichard Josette , Cramer Elisabeth M. , Halbwachs-Mecarelli Lise
Published: April 2021

We investigated a possible association of leukosialin (CD43), the major surface sialoglycoprotein of leukocytes, with neutrophil cytoskeleton. We first analysed the solubility of CD43 in Triton X-100 and observed that CD43 of resting neutrophils was mostly soluble. The small proportion of CD43 molecules, which ‘spontaneously’ precipitated in Triton, appeared associated with F-actin, as demonstrated by the fact that this insolubility did not occur when cells were incubated with cytochalasin B or when F-actin was depolymerized with DNase I in the Triton precipitate. Cell stimulation with anti-CD43 mAb (MEM59) enhanced this CD43-cytoskeleton association. By immunofluorescence as well as by electron microscopy, we observed a redistribution of CD43 on the neutrophil membrane, initially in patches followed by caps, during anti-CD43 cross-linking at 37°C. This capping did not occur at 4°C and was inhibited by cytochalasin B and by a myosin disrupting drug butanedione monoxime, thus providing evidence that the actomyosin contracile sytem is involved in the capping and further suggesting an association of CD43 with the cytoskeleton. Some of the capped cells exhibited a front-tail polarization with CD43 caps located in the uropod at the rear of the cell. Surprisingly, colchicine and the chemotactic factor fNLPNTL which induce neutrophil polarization associated with cell motility, also resulted in a clustering of CD43 in the uropod, independently of a cross-linking of the molecule by mAbs. An intracellular redistribution of F-actin, mainly at the leading front and of myosin in the tail, was observed during CD43 clustering induced by colchicine and in cells polarized by anti-CD43 mAbs cross-linking. We conclude that neutrophil CD43 interacts with the cytoskeleton, either directly or indirectly, to redistribute in the cell uropod under antibodies stimulation or during cell polarization by colchicine, thus highly suggesting that CD43 may be involved in cell polarization.
Evidence for colocalization and interaction between 37 and 39 kDa isoforms of secretory carrier membrane proteins (SCAMPs)
Authors: Wu Theodore T. , Castle J. David
Published: April 2021

Secretory carrier membrane proteins (SCAMPs) are proteins of post-Golgi recycling carriers, including regulated secretory organelles. The two major size variants, SCAMP1 (37 kDa) and SCAMP2 (39 kDa), extensively colocalize in membranes of fibroblasts and parotid acinar cells based on immunocytochemistry and velocity centrifugation, although the relative amounts of each variant may differ in selected organelles. SCAMP1, and to a lesser extent, SCAMP2, are substrates for chemical crosslinking in situ, and the recognizable crosslinking products of SCAMP1 suggest potential formation of homomultimers. SCAMP1 and SCAMP2 can be co-immunoprecipitated following detergent solubilization, using antibodies that specifically react with only one of the variants. Both the localization and interactions of SCAMPs are reiterated using transfected SCAMP1 that is epitope tagged (myc) at either the NH2 or COOH terminus and an anti-myc antibody. Like other transport vesicle membrane proteins, SCAMPs form complexes that apparently include homomultimers. Furthermore, these studies suggest that both SCAMP1 and SCAMP2 may function together in a single protein complex.
Novel αGalNAc containing glycans on cytokeratins are recognized in vitro by galectins with type II carbohydrate recognition domains
Authors: Goletz Steffen , Hanisch Franz-Georg , Karsten Uwe
Published: April 2021

We report on a novel posttranslational modification of cytoplasmic proteins. Presented evidences suggest that cytokeratins are bound in vitro by mammalian galectin-3 and the galectins from the sponge Geodia cydonium via their type II carbohydrate recognition domains, whose highest binding affinity is directed towards terminal α-N-acetylgalactosamine-bearing glycans with the general sequence GalNAcα1-3Gal(NAc) β. Specificity analyses and the characterization of the critical sugar residue on cytokeratins for galectin binding were done with cytochemical and biochemical methods using various plant and animal lectins. Binding of GalNAc-specific lectins was saturable, sensitive to mild periodate oxidation, inhibitable by glycoconjugates carrying terminal GalNAc, and abolished after treatment of the cytokeratins with α-N-acetylgalactosaminidase. Binding to bacterially expressed recombinant cytokeratins did not exceed background binding. The presence of GalNAc residues on highly purified cytokeratins from MCF-7 and HeLa SS6 cells was confirmed by sugar composition analyses using gas chromatography/mass spectrometry. This novel posttranslational modification was not restricted to cytokeratins of MCF-7 cells, but did also occur in all of 9 other examined human carcinoma cell lines and in a normal human mammary epithelial cell line. From these cytochemical and biochemical in vitro studies we hypothesize that this glycan with its terminal α1-3 linked GalNAc determinant might represent the first natural cytoplasmic ligand for endogenous galectins-3 detected so far.
Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa
Authors: Westhoff Doris , Kamp Günter
Published: April 2021

Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin (15 minutes) even resulted in an activation of glyceraldehyde 3-phosphate dehydrogenase. Purification on phenyl-Sepharose yielded a homogeneous glyceraldehyde 3-phosphate dehydrogenase as judged from gel electrophoresis SDS-PAGE and native gradient PAGE. The molecular masses are 41.5 and 238 kDa, respectively, suggesting native glyceraldehyde 3phosphate dehydrogenase to be a hexamer.

Rabbit polyclonal antibodies raised to purified glycer-aldehyde 3-phosphate dehydrogenase show a high specificity for mammalian spermatozoal glyceraldehyde 3phosphate dehydrogenase, while other proteins of boar spermatozoa or the muscle glyceraldehyde 3-phosphate dehydrogenase are not labelled. Immunogold staining performed in a post-embedding procedure reveals the localization of glyceraldehyde 3-phosphate dehydrogenase along the fibrous sheath in spermatozoa of boar, bull, rat, stallion and man. Other structures such as the cell membrane, dense fibres, the axoneme or the mitochondria are free of label.

During the process of sperm maturation, most of the cytoplasm of the sperm midpiece is removed as droplets during the passage through the epididymis. The labelling of this cytoplasm, in immature boar spermatozoa and in the droplets, indicates that glyceraldehyde 3-phosphate dehydrogenase is completely removed from the midpiece during sperm maturation in the epididymis.

The inverse compartmentation of the glycolytic enzyme and mitochondria in the mammalian sperm flagella suggests that ATP-production in the principal piece mainly occurs by glycolysis and in the midpiece by respiration.
Twisted scroll waves organize Dictyostelium mucoroides slugs
Authors: Dormann Dirk , Weijer Cornelis , Siegert Florian
Published: April 2021

Cellular slime moulds (Dictyosteloids) are characterised by at least two different modes of slug migration. Most species, e.g. Dictyostelium mucoroides, produce a stalk continuously during slug migration, while a few species, e.g. Dictyostelium discoideum are characterised by stalk-less slug migration and only produce a stalk upon culmination. Experiments on D. discoideum and theoretical model calculations have shown that D. discoideum slugs are organized by a cAMP scroll wave in the tip which produces planar waves in the back. These waves guide cell movement in slugs: spiralling in the tip and forward movement parallel to the slug axis in the back. Simple changes in model parameters can lead to the formation of a twisted scroll wave which extends throughout the slug. In order to investigate whether such twisted scroll waves occur naturally we have analysed the movement of fluorescently labelled single cells in migrating D. mucoroides slugs. The results show that cells in the prespore zone of D. mucoroides slugs move in a spiral path. Although the velocity of single cells in D. mucoroides is faster than in D. discoideum, the net forward component of their movement is less due to their spiral trajectories. As a result D. mucoroides slugs move more slowly than D. discoideum slugs. The entire D. mucoroides slug also describes a spiralling path leaving corkscrew shaped stalks behind. Based on these observations we propose that cell movement in D. mucoroides slugs is controlled by a propagating twisted scroll wave of cAMP which extends throughout the length of the slug.
Megalomicin disrupts lysosomal functions
Authors: Bonay Pedro , Fresno Manuel , Alarcón Balbino
Published: April 2021

Megalomicin (MGM) has been shown to cause a dilation of the most distal cisternae of the Golgi complex. The effects of MGM on Golgi morphology correlated with an inhibition of protein transport to the trans-Golgi resulting in an accumulation of poorly sialylated glycoproteins. Here we show that the addition of 50 μM MGM caused a rapid swelling of lysosomes in cultured cells and inhibited the degradation of the newly synthesized T cell antigen receptor CD3δ subunit. Although MGM did not affect the uptake of fluid phase markers, it prevented their degradation. Interestingly, endocytosed ovalbumin did not colocalize with lysosomes in MGM-treated cells, suggesting an MGM-induced impairment in the delivery to lysosomes. This was confirmed by Percoll density gradients, where the fluid phase marker remained in endosomal fractions, even after long chase times, whereas in control cells the endocytosed marker was located in lysosomes. The effect of MGM was not confined to soluble proteins since it did also inhibit the delivery of the membrane-bound epidermal growth factor receptor to lysosomes. Finally, MGM strongly inhibited the ATP-dependent acidification of lysosomes in vitro, suggesting a possible mechanism for its in vivo activity.
Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe
Authors: Hagan Iain , Yanagida Mitsuhiro
Published: April 2021

Specific changes in spatial order occur during cell cycle progression in fission yeast. Growth of the rod-shaped cells is highly regulated and undergoes a cell cycle and sizeregulated switch from monopolar to bipolar tip extension. During both phases of growth, the interphase nucleus is maintained in a central location. Following the separation of the genome to the cell tips in mitosis, the two nuclei migrate back towards the cell equator before stopping in two new positions that will become the middle of the two new cells. Here we use simultaneous labeling of microtubules, chromatin and spindle pole bodies in wild-type and cdc mutants, to show that nuclear positioning is achieved by regulation of spindle pole body-mediated nuclear migration. We show that the number and location of nuclear positioning signals is regulated in a cell cyclespecific manner and that spindle pole body-mediated forces are likely to be responsible for maintaining correct nuclear position once the nuclei have reached the appropriate position in the cell. Accentuating the movement of the nuclei back towards the cell equator after mitosis by artificially increasing cell length shows that the spindle pole body leads the nucleus during this migration. When multiple spindle pole bodies are associated with the same or different nuclei they all go to the same point indicating that the different spindle pole bodies are responding to the same positional cue. In a septation-defective mutant cell, which contains four nuclei, the spindle pole bodies on the four different nuclei initially group as two pairs in regions that would become the middle of the new cells, were the cell able to divide. In the subsequent interphase, the nuclei aggregate as a group of four in the centre of the cell. The presence of two or three clusters of spindle pole bodies in larger cells with eight nuclei suggests that the mechanisms specifying the normally central location for multiple nuclei may be unable to operate properly as the cells get larger. Perturbation of microtubules with the microtubule poison thiabendazole prevents the spindle pole body clustering in septation mutants, demonstrating that nuclear positioning requires a functional microtubule cytoskeleton.
Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells
Authors: Lampugnani Maria Grazia , Corada Monica , Andriopoulou Paraskevi , Esser Sybille , Risau Werner , Dejana Elisabetta
Published: April 2021

In srcand ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, β-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with β-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and β-catenin more efficiently, but not plakoglobin.

Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for β-catenin binding, and PV stabilized this association.

Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and β-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.
Insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-κB transcription factor
Authors: Benoliel Anne-Marie , Kahn-Perles Brigitte , Imbert Jean , Verrando Patrick
Published: April 2021

Insulin-mediated cell motility as well as the role of transcription factors in insulin-activated intracellular signal events have not been extensively studied. In this report we have examined whether insulin could mediate haptotactic migration of cultured human epidermal keratinocytes through activation of transcription factor NF-κB. Insulin caused a dose-dependent stimulation of keratinocyte migration that maximally reached 2-fold at 2×10−7 M hormone. This phenomenon was independent of the nature of the extracellular matrix component (collagen I or laminin5/nicein) on which the cells migrated, indicating that a specific integrin-ligand complex is not required. A 10−7 M insulin treatment of keratinocytes resulted in activation of a major κB DNA binding complex within 15 to 30 minutes, which was identified as the p65/p50 NF-κB heterodimer by electrophoretic mobility shift assays. The activation induced nuclear translocation of cytosolic pools of NF-κB factor. Pyrrolidine dithiocarbamate and N-acetylleucinyl-leucinyl-norleucinal H (two compounds that differentially inhibit IκBα degradation and, thus, NF-κB activation) reversed the insulin-stimulated keratinocyte haptotactic migration without affecting insulin receptor activation. These compounds inhibited the insulin-induced nuclear translocation of NF-κB as detected by confocal laser scanning microscopy. Taken together our experiments demonstrate that insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-κB transcription factor. They emphasize the ability of insulin to stimulate keratinocyte movement and provide a first clue to the mechanism of insulin-induced haptotactic signaling.
Association of host cell endoplasmic reticulum and mitochondria with theToxoplasma gondiiparasitophorous vacuole membrane: A high affinity interaction
Authors: Sinai Anthony P. , Webster Paul , Joiner Keith A.
Published: April 2021

The parasitophorous vacuole membrane (PVM) of the obligate intracellular protozoan parasite Toxoplasma gondii forms tight associations with host mitochondria and the endoplasmic reticulum (ER). We have used a combination of morphometric and biochemical approaches to characterize this unique phenomenon, which we term PVM-organelle association. The PVM is separated from associated mitochondria and ER by a mean distance of 12 and 18 nm, respectively. The establishment of PVM-organelle association is dependent on active parasite entry, but does not require parasite viability for its maintenance. Association is not a consequence of spatial constraints imposed on the growing vacuole. Morphometric analysis indicates that the extent of mitochondrial association with the PVM stays constant as the vacuole enlarges, whereas the extent of ER association decreases. Disruption of host cell microtubules partially blocks the establishment but not the maintenance of PVM-mitochondrial association, and has no significant effect on PVM-ER association. PVM-organelle association is maintained following disruption of infected host cells, as assessed by electron microscopy and by sub-cellular fractionation showing co-migration of fixed PVM and organelle markers. Taken together, the data suggest that a high affinity, potentially protein-protein interaction between parasite and organelle components is responsible for PVM-organelle association.
Nuclear envelope breakdown in mammalian cells involves stepwise lamina disassembly and microtubule-driven deformation of the nuclear membrane
Authors: Georgatos Spyros D. , Pyrpasopoulou Athina , Theodoropoulos Panayiotis A.
Published: April 2021

We have studied nuclear envelope disassembly in mammalian cells by morphological methods. The first signs of nuclear lamina depolymerization become evident in early prophase as A-type lamins start dissociating from the nuclear lamina and diffuse into the nucleoplasm. While B-type lamins are still associated with the inner nuclear membrane, two symmetrical indentations develop on antidiametric sites of the nuclear envelope. These indentations accommodate the sister centrosomes and associated astral microtubules. At midto late prophase, elongating microtubules apparently push on the nuclear surface and eventually penetrate the nucleus. At this point the nuclear envelope becomes freely permeable to large ligands, as indicated by experiments with digitonin-treated cells and by the massive release of solubilized A-type lamins into the cytoplasm. At the prophase/prometaphase transition, the B-type lamina is fragmented, but ‘islands’ of lamin B polymer can still be discerned on the tips of congressing chromosomes. Finally, at metaphase, the lamin B polymer breaks down into small pieces, which tend to concentrate in the area of the mitotic spindle. Nuclear envelope breakdown is not prevented when the microtubules are depolymerized by nocodazole; however, the mode of nuclear lamina fragmentation in the absence of microtubules is markedly different from the normal one and involves multiple raffles and gaps, which develop rapidly along the entire surface of the nuclear envelope. These data suggest that nuclear envelope disassembly is a stepwise process in which the microtubules play an important part.
Myosin II is associated with Golgi membranes: identification of p200 as nonmuscle myosin II on Golgi-derived vesicles
Authors: Ikonen Elina , de Almeid J. Bruno , Fath Karl R. , Burgess David R. , Ashman Keith , Simons Kai , Stow Jennifer L.
Published: April 2021

A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells. A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated. Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II. We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes. Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles. The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins. Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA). Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin. These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.
Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in FcεRI-activated rat basophilic leukemia (RBL-2H3) cells
Authors: Guillemot Jean-Claude , Montcourrier Philippe , Vivier Eric , Davoust Jean , Chavrier Philippe
Published: April 2021

Engagement of the high affinity IgE receptor (FcεRI) in mast cells elicits a series of intracellular signalling events including cytoskeletal reorganization and granule exocytosis. To analyze the coupling of receptor activation to specific cytoskeletal responses, we expressed dominant negative mutant forms of the Rho GTPases CDC42 and Rac1 in rat RBL-2H3 tumor mast cells. We show here that dominant inhibition of CDC42 function decreases cell adhesion, interferes with FcεRI-induced actin plaque assembly and reduced the recruitment of vinculin at the cell-substratum interface, while the inhibitory Rac1 mutant abolishes FcεRI-mediated membrane ruffling. The expression of trans-dominant inhibitory forms of either CDC42 or Rac1 significantly inhibited antigen-induced degranulation. Altogether, our results demonstrate that CDC42 and Rac1 control distinct pathways downstream of FcεRI engagement leading either to the induction of actin plaques, or to the production of membrane ruffles. These two pathways are critically involved during the degranulation response induced by FcεRI aggregation.
Dictyostelium lysosomal proteins with different sugar modifications sort to functionally distinct compartments
Authors: Souza Glaucia M. , Mehta Darshini P. , Lammertz Marion , Rodriguez-Paris Juan , Wu Rongrong , Cardelli James A. , Freeze Hudson H.
Published: April 2021

Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P-and Man-6-P-bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohy-drate modifications probably establishes and/or maintains segregation.
The glucose transporter GLUT4 and the aminopeptidase vp165 colocalise in tubulo-vesicular elements in adipocytes and cardiomyocytes
Authors: Martin Sally , Rice Jacqueline E. , Gould Gwyn W. , Keller Susanna R. , Slot Jan W. , James David E.
Published: April 2021

The aminopeptidase vp165 is one of the major polypeptides enriched in GLUT4-containing vesicles immuno-isolated from adipocytes. In the present study we have confirmed and quantified the high degree of colocalisation between GLUT4 and vp165 using double label immuno-electron microscopy on vesicles isolated from adipocytes and heart. The percentage of vp165-containing vesicles that also contained GLUT4 was 91%, 76%, and 86% in rat adipocytes, 3T3-L1 adipocytes, and rat heart, respectively. Internalisation of a transferrin/HRP (Tf/HRP) conjugate by 3T3-L1 adipocytes, followed by diaminobenzidine treatment in intact cells, resulted in ablation of only 41% and 45% of GLUT4 and vp165, respectively, whereas endosomal markers are almost quantitatively ablated. Using immuno-electron microscopy on cryosections it was determined that in atrial cardiomyocytes GLUT4 and vp165 colocalised in a population of tubulo-vesicular (T-V) elements that were often found close to the plasma membrane. Double label immunocytochemistry indicated a high degree of overlap in these T-V elements between GLUT4 and vp165. However, in atrial cardiomyocytes a large proportion of GLUT4 was also present in secretory granules containing atrial natriuretic factor (ANF). In contrast, very little vp165 was detected in ANF granules. These data indicate that GLUT4 and vp165 are colocalised in an intracellular, post-endocytic, tubulo-vesicular compartment in adipocytes and cardiomyocytes suggesting that both proteins are sorted in a similar manner in these cells. However, GLUT4 but not vp165 is additionally localised in the regulated secretory pathway in atrial cardiomyocytes.
Differential induction of apoptosis in Swiss 3T3 cells by nitric oxide and the nitrosonium cation
Authors: Khan Shazia , Kayahara Midori , Joashi Umesh , Mazarakis Nicholas D. , Sarraf Catherine , Edwards A. David , Hughes Martin N. , Mehmet Huseyin
Published: April 2021

We have investigated the effect of nitric oxide (NO) on apoptosis in Swiss 3T3 fibroblasts and compared it to the effect of the nitrosonium cation (NO+). Both species induced apoptosis, confirmed by electron microscopy, propidium iodide staining, DNA laddering and activation of caspases. The kinetics of triggering apoptosis were different for the two redox species: NO+ required only a 2 hour exposure, whereas NO required 24 hours. Three sources of NO were used: aqueous solutions of NO and two NO donors, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine. The time course of apoptosis induced by these two S-nitrosothiols correlated with their rate of decomposition to NO. The apoptotic effect of NO was reduced in the presence of the NO scavenger oxyhaemoglobin, or the antioxidants N-acetylcysteine and ascorbic acid, whereas in the case of NO+ these antioxidants potentiated apoptosis. Glutathione also had a potentiating effect on the cytotoxicity of NO+. This suggests that cellular antioxidants may play a role in protecting the cell from NO-induced apoptosis while NO+ may trigger apoptosis independently of oxidative stress mechanisms.